Immunomodulatory polymeric antigens for treating inflammatory pathologies

ABSTRACT

Provided are natural and synthetic immunomodulatory polymeric antigens (SPAs), compositions containing SPAs, and methods of using these natural and synthetic SPAs and compositions to prevent or treat inflammatory pathologies.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of immunology, and moreparticularly to immunomodulation. The present invention provides novelmethods for preventing and treating inflammatory pathologies employingnatural or synthetic polymeric antigens (N/S PAs) possessingimmunomodulatory properties in these methods. The present invention alsoprovides a process for preparing novel synthetic SPAs that can be usedto induce the activity of T regulatory cells and the expression ofinterleukin 10 (IL10) in humans and other animals, affording protectionagainst, and/or treatment for, a wide variety of inflammation-basedpathologies.

2. Description of Related Art

Microbial antigens are the most powerful immunomodulators known. Amongthe most common examples are lipopolysaccharide (LPS) from Gram negativebacteria, and bacterial cell wall glycopeptides, also known as murein orpeptidoglycan (PG), from both Gram negative and Gram positive bacteria.Bacterial PG is well established as a potent inflammatory agent (Wahl etal. (1986) J. Exp. Med. 165:884). Many microbial antigens, including PG,are thought to exert their pro-inflammatory effects by activating one ofthe mammalian cell surface receptors known as Toll-like receptors(TLRs). The TLR then triggers an intracellular signaling pathway throughtranscription factor NF-κB, which in turn induces expression of genescoding for inflammatory mediators (chemokines and certain cytokines). PGitself is thought to activate through TLR2 (Hallman et al. (2001)Pediatr. Res. 50:315).

Recently, cDNA array technology has brought even higher resolution toour understanding of pro-inflammatory mediator induction by PG (Wang etal. (2000) J. Biol. Chem. 275:20260). The most highly activated genesare those expressing chemokines (IL-8 and MIP-1β), and the second mosthighly activated genes are those expressing cytokines (TNF-α, IL1, andIL6). Regardless of mechanistic detail, the downstream effect ofbacterial PG on the host is a potent inflammatory response. In fact, PGhas long been used for induction of arthritis in animal models(Cromartie et al. (1977) J. Exp. Med. 146:1585). Partially purified PGfrom the bacterium Streptococcus pyogenes is now commercially availablefor such purpose (Lee Laboratories, Atlanta, Ga.). Fragments of PG,known collectively as muropeptides, also exhibit inflammatory effects inanimals, and these effects are dependent on muropeptide structure(Tuomanen et al. (1993) J. Clin. Invest. 92:297). Even the very smallestfragments of PG, designated muramyl dipeptide (MDP), and glucosaminylMDP (GMDP), as well as their derivatives, exhibit inflammatory effectsin animals (Kohashi et al. (1980) Infect. Immun. 29:70).

Kasper and Tzianabos have demonstrated that certain polysaccharidespurified from the surface of bacterial cells exhibit protective effectsin vivo when tested in models of inflammation such as the formation ofintraabdominal abscesses, intraabdominal sepsis, and post-surgicaladhesions (U.S. Pat. Nos. 5,679,654 and 5,700,787; PCT InternationalPublications WO 96/07427, WO 00/59515, and WO 02/45708). Theseinvestigators have demonstrated that when purified from whole capsule,certain polysaccharides derived from Bacteroides fragilis,Staphylococcus aureus, and Streptococcus pneumoniae have uniquecharacteristics that set them apart from many polysaccharide antigens.The former molecules are high molecular weight, helical, andzwitterionic in nature (Wang et al. (2000) Proc. Natl. Acad. Sci. USA97:13478-13481, and references 5-9 therein). Most bacterialpolysaccharides are neutral or negatively charged, and are considered tobe T cell-independent antigens (Abbas et al. (2000) Cellular andMolecular Immunobiology, W.B. Saunders, Philadelphia). Kasper andTzianabos suggest that the zwitterionic nature of these polysaccharidesplays a role in the interaction of these molecules with CD4+ T cells(Tzianabos et al. (1993) Science 262: 416-419; Tzianabos et al. (2001)Proc. Natl. Acad. Sci. USA 98:9365-9370). More recent work by this groupsuggests that some of these molecules may interact with antigenpresenting cells (APCs) via their zwitterionic characteristics andfurther, that stimulation of CD4+ T cells by these polysaccharideantigens is dependent on MHC II-bearing APCs (Kalka-Moll et al. (2002)J. Immunol. 169:6149-6153). It has yet to be determined precisely howthese interactions between zwitterionic polysaccharides and APCs maystimulate CD4+ T cells. These investigators have shown that zwitterionicpolysaccharides activate CD4+ T cells in vitro as evidenced by thestimulation of proliferation and the production of the cytokines IL2,INFγ, and IL10, and that the protection is adoptively transferred bypolysaccharide-stimulated T cells in vivo (PCT International PublicationWO 00/59515; Kalka-Moll et al. (2000) J. Immunol. 164:719-724; Tzianaboset al. (2000) J. Biol. Chem. 275:6733-6738). In earlier studies by thisgroup, stimulation of CD4+ cells did not necessarily depend on thepresence of APCs, and the mitogenic properties of these molecules on Tcells derived from rat and mouse species was different: rat splenocytesproliferated in response to CP1 treatment, while mouse splencocytes didnot (Tzianabos et al. (1995) J. Clin. Invest. 96:2727-2731; Brubaker etal. (1999) J. Immunol. 162:2235-2242).

Overall, however, their observations led this group to hypothesize thatthe activation of CD4+ T cells by these polysaccharides leads to theproduction of cytokines such as IL2 or IL10 that protect againstinflammatory responses (PCT International Publication WO 00/59515;Kalka-Moll et al. (2000) J. Immunol. 164:719-724; Tzianabos et al.(2000) J. Biol. Chem. 275:6733-6738; Tzianabos et al. (1999) J. Immunol.163: 893-897). It remains unclear, however, exactly how these moleculesactivate T cells or how they exert their protective effects. Furthercomplicating an understanding of these polysaccharides, this group hasreported other studies indicating that the same zwitterionicpolysaccharides can induce the formation of abscesses in the same invivo model where protective effects of these molecules have beenobserved (Tzianabos et al. (1993) Science 262: 416-419; Tzianabos et al.(1994) Infect. Immun. 62:3590-3593). Therefore, from this body ofliterature, it is difficult to ascertain the mechanism whereby thesezwitterionic polysaccharides act as modulators of the immune system.

Another group of investigators has described immunomodulatory effects ofthe exopolysaccharide (capsule-like) of Paenibacillus jamilae, a grampositive bacillus isolated from olive mill wastewaters (Ruiz-Bravo etal. (2001) Clin. Diag. Lab. Immunol. 8:706-710). Although the authors donot disclose the structural features of this polysaccharide, theirresults are similar to the work of Kasper and Tzianabos, summarizedabove. The molecule, referred to as CP-7, stimulates the proliferationof lymphocytes in culture, as well as significant expression of IFNγ andGMCSF. Further, this group reports that this compound renders miceresistant to Listeria monocytogenes infection. The investigators suggestthat the mechanism may be through the stimulation of a Th1 response,which is in direct contrast to the invention disclosed herein.

In view of the confusing and sometimes contradictory effects reported inthe literature for various immunomodulatory polysaccharides, thereexists a need in the art for an understanding of the mechanism of actionof protective, anti-inflammatory immunomodulatory molecules, includingpolysaccharides, as well as a need for additional therapeutic moleculesthat modulate the immune response in both a safe and effective manner.Such insight and additional molecules will facilitate the development ofeven more effective anti-inflammatory strategies and therapeutics.

SUMMARY OF THE INVENTION

Accordingly, in view of the need in the art for an understanding of themechanism(s) by which immunomodulatory polysaccharide antigens induceprotection against inflammation, as well as the need for additionalmolecules that can be used to modulate the immune response in humans andanimals for anti-inflammatory therapeutic purposes, the presentinventors have investigated the properties and effects of animmunomodulatory bacterial polysaccharide and a novel syntheticpeptidoglycan on immune system function. They have discovered that thebacterial polysaccharide derived from the capsule of Streptococcuspneumoniae, referred to as CP1, as well as the novel syntheticpeptidoglycan (PG) Compound 15 disclosed herein, which is a syntheticpolymeric antigen, protect against the induction of inflammation inmodels of intraabdominal abscesses and post-surgical adhesions. Theyhave also surprisingly discovered that when human peripheral bloodmononuclear cells (PBMCs) are treated in vitro with an SPA as disclosedherein, the response is most notably the expression of IL10. Onlyminimal and early expression of IL2, IFN-γ, or TNF-α is observed. Thestimulation of an anti-inflammatory response by the syntheticpeptidoglycan polymer disclosed herein is completely novel andunexpected in view of the current body of evidence discussed above:while natural peptidoglycans are inflammatory, the presently disclosedsynthetic peptidoglycan is anti-inflammatory. The inventors' surprisingdiscovery of the in vitro anti-inflammatory activity of an SPA contrastsmarkedly with previously published observations on the activity ofpurified bacterial surface polysaccharides, and prompted them to testthe activity of this SPA in an animal model of inflammation. Theinventors observed that this SPA exhibits protective therapeutic effectsin this animal model of inflammation-based pathology.

Accordingly, in one aspect, the present invention provides a syntheticpolymeric antigen having the structure shown in Formula I:

-   -   wherein n is an integral in the range of from about 375 to about        75,    -   or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a composition,comprising the synthetic polymeric antigen or pharmaceuticallyacceptable salt thereof of Formula I, and a buffer, carrier, diluent, orexcipient.

In another aspect, the present invention provides a pharmaceuticalcomposition, comprising the synthetic polymeric antigen orpharmaceutically acceptable salt thereof of Formula I, and apharmaceutically acceptable buffer, carrier, diluent, or excipient.

In another aspect, the present invention provides a method of inhibitingthe maturation of an antigen presenting cell, comprising contacting invitro said antigen presenting cell and an effective amount of a compoundselected from the group consisting of CP1, Compound 15, and mixturesthereof, for a time and under conditions effective to inhibit maturationof said antigen presenting cell.

In another aspect, the present invention provides a method of inhibitingthe maturation of an antigen presenting cell in a mammal, comprisingadministering to a mammal other than a rat or a mouse an effectiveamount of a compound selected from the group consisting of CP1, Compound15, and mixtures thereof, and inhibiting maturation of said antigenpresenting cell.

In another aspect, the present invention provides a method of inhibitingan inflammatory response in a mammal in need thereof other than a rat ora mouse, comprising:

-   -   (a) isolating peripheral blood mononuclear cells, or a        monocyte-containing fraction thereof, from said mammal;    -   (b) contacting in vitro said isolated peripheral blood        mononuclear cells or monocytes and a composition containing an        effective amount of cytokines that differentiate monocytes to        immature dendritic cells for a time and under conditions        effective to generate immature monocyte-derived dendritic cells;    -   (c) contacting in vitro said immature monocyte-derived dendritic        cells and an effective amount of a compound selected from the        group consisting of CP1, Compound 15, and a mixture thereof for        a time and under conditions effective to prevent maturation of        said immature monocyte-derived dendritic cells; and    -   (d) administering said immature monocyte-derived dendritic cells        to said mammal, reducing the ability of dendritic cells of said        mammal to drive cognate interactions with T cells and inhibiting        said inflammatory response in said mammal.

In this and the other ex vivo methods disclosed herein, administrationof treated cells can be performed intravenously, intraperitoneally, orvia intercardiac route.

Inflammatory responses that can be treated via the foregoing andfollowing methods include abscesses and post-surgical adhesions, sepsis;rheumatoid arthritis; myesthenia gravis; inflammatory bowel disease;colitis; systemic lupus erythematosis; multiple sclerosis; coronaryartery disease; diabetes; hepatic fibrosis; psoriasis; eczema; acuterespiratory distress syndrome; acute inflammatory pancreatitis;endoscopic retrograde cholangiopancreatography-induced pancreatitis;burns; atherogenesis of coronary, cerebral, and peripheral arteries;appendicitis; cholecystitis; diverticulitis; visceral fibroticdisorders; wound healing; skin scarring disorders; granulomatousdisorders; asthma; pyoderma gangrenosum; Sweet's syndrome; Behcet'sdisease; primary sclerosing cholangitis; and cell, tissue, or organtransplantation.

In yet another aspect, the present invention provides a method ofinhibiting an inflammatory response in a mammal in need thereof otherthan a rat or a mouse, comprising:

-   -   administering to said mammal an effective amount of a compound        selected from the group consisting of CP1, Compound 15, and        mixtures thereof, preventing dendritic cells or other antigen        presenting cells of said mammal from maturing and rendering them        incapable of stimulating T cell activation,    -   thereby inhibiting said inflammatory response in said mammal.

In another aspect, the present invention provides a method of inhibitingan inflammatory response in a mammal in need thereof other than a rat ora mouse, comprising:

-   -   (a) isolating peripheral blood mononuclear cells, or a        monocyte-containing fraction thereof, from said mammal;    -   (b) contacting in vitro said isolated peripheral blood        mononuclear cells or monocytes and a composition containing an        effective amount of cytokines that differentiate monocytes to        immature dendritic cells for a time and under conditions        effective to generate immature monocyte-derived dendritic cells;    -   (c) contacting in vitro said immature monocyte-derived dendritic        cells and an effective amount of a compound selected from the        group consisting of CP1, Compound 15, and mixtures thereof for a        time and under conditions effective to prevent maturation of        said immature monocyte-derived dendritic cells;    -   (d) contacting in vitro said immature dendritic cells and naïve        T cells to generate T regulatory cells; and    -   (e) administering said T regulatory cells that suppress T        effector cells to said mammal,        -   thereby suppressing said inflammatory response.

In a further aspect, the present invention provides a method ofinhibiting an inflammatory response in a mammal in need thereof otherthan a rat or a mouse, comprising:

-   -   administering to said mammal an effective amount of a compound        selected from the group consisting of CP1, Compound 15, and        mixtures thereof,    -   generating T regulatory cells that suppress T effector cells and        that inhibit said inflammatory response.

In another aspect, the present invention provides a method of measuringthe immunological activity of CP1 or Compound 15 in a mammal,comprising:

-   -   administering CP1 or Compound 15 to said mammal;    -   administering Candin to said mammal; and    -   measuring the inhibition of delayed type hypersensitivity skin        lesions elicited by said Candin,    -   wherein a reduction in lesion size in said mammal compared to        lesion size in an untreated control mammal that has not received        CP1 or Compound 15 indicates that said compounds are effective        in inhibiting a localized inflammatory response.

Further scope of the applicability of the present invention will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the presentinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 is a schematic showing the normal events that occur wheninteractions between dendritic cells and T cells lead to inflammation oradaptive immunity

FIG. 2 is a schematic showing the T regulatory cell hypothesis of thepresent invention.

FIG. 3 shows the cytokine profile from human peripheral bloodmononuclear cells (PBMCs) treated with CP1 or Compound 15 (PG). HumanPBMCs in culture are treated with CP1 at 6.0 micrograms/ml (panel A) orPG at 0.6 micrograms/ml (panel B), and the expression of cytokines ismeasured over the course of eight days. Results are normalized againstuntreated media controls. Data are expressed as the average oftriplicate wells 3± the standard error of the concentration of cytokinesrepresented. The results show that the primary response to treatmentwith these molecules is the expression of IL10.

FIG. 4 shows Confocal microscope images of human iDCs treated witheither FITC-Dextran (FITC-Dx, 40 kDa in size) or Oregon-green labeledCompound 15 (OG-PG, approx. 150 kDa in size) for two minutes. Afterincubation with the polymers, the cells are washed extensively to removeany external polymer and the internalized material followed attwo-minute intervals. Localization of polymer in endocytic vacuoles canbe seen using either compound, and fluorescence is visualized in thephotographs as white punctate material within the dark field of thecells.

FIG. 5 shows flow cytometric analysis of uptake of either FITC-Dextran(panel A) or Oregon-green labeled Compound 15 (panel B) by human DC at37° C. or 0° C., respectively. Each histogram shows the meanfluorescence intensity of fluorescent signal versus cell number at thetime intervals indicated. The results show that the uptake of eachmolecule is similar, and that this uptake is inhibited when the cellsare metabolically inactive at 0° C.

FIG. 6 shows that neither CP1 nor PG induces PBMCs to divide in culture.Isolated PBMCs are incubated with 50 μg/ml CP1 (●), 100 μg/ml pg (◯), 25μg/ml PHA (∇), or left untreated (▾) for the number of days indicated.Radioactive thymidine [³H]-Thy is added to cultures 18 h prior to eachtime point and the amount of radiolabel incorporated by the cells ismeasured by scintillation counting. Radioactivity is measured as countsper minute.

FIG. 7 shows that CP 1 induces an increase in the percent of CD4+CD25+ Tregulatory cells in human PBMCs in a dose-dependent manner. IsolatedPBMCs are incubated with CP1 at 0.6 micrograms/ml (closed triangle) or6.0 micrograms/ml (closed square) for the number of days indicated.Untreated PBMCs (closed circle) are included as a measure of the baseline number of CD4+CD25+ cells present in the culture.

FIG. 8 shows that CP1 and synthetic PG Compound 15 inhibit anti-CD3antibody-mediated proliferation of human PBMCs. PBMCs are pre-incubatedfor 24 hours with 50 mg/ml of CP 1 or 100 mg/ml of PG Compound 15 priorto incubation on tissue culture plates coated with varyingconcentrations of anti-CD3 antibody for 48 hours (panel A) or 72 hours(panel B). Cell proliferation is evaluated using a ³H-Thymidineincorporation assay followed by liquid scintillation counting.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the in practicing the present invention. Even so, thefollowing detailed description should not be construed to unduly limitthe present invention as modifications and variations in the embodimentsdiscussed herein can be made by those of ordinary skill in the artwithout departing from the spirit or scope of the present inventivediscovery.

The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

DEFINITIONS

As used herein, unless indicated otherwise, the following abbreviationsshall be understood to have the following meanings:

Reagent or Abbreviation Fragment h or hr hour(s) min. minute(s)

As used above, and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

“Biomarker” means a marker of a specific activity that correlates withthe administration of a drug. Non-limiting examples of biomarkersinclude a cell surface receptor, a soluble mediator, an mRNA message, oran in vivo response that is modulated and that can be measured.

“CP1” means a capsular polysaccharide obtainable from Streptococcuspneumoniae serotype 1. CP1 is used in the methods disclosed herein in aform isolated and purified as described below.

“Effective amount” refers to an amount of a compound or composition ofthe present invention effective to produce the desired or indicatedimmunologic or therapeutic effect.

“IL10” is an endogenous mediator that shifts equilibrium away frominflammation. Directed, endogenous generation of IL10 maximizes efficacyand minimizes toxic effects.

“Immune cell” means any cell capable of responding or mounting aresponse within the entirety of the host immune system. Generally thesecells are referred to as “white blood cells” but are not necessarilylimited to this category. Examples of immune cells include T and Bcells, monocytes, macrophages, natural killer cells, dendritic cells,antigen presenting cells, and polymorphonuclear leukocytes.

“Modulate” means either an increase or a decrease in a selectedparameter.

The terms “patient” or “subject” refer to mammals including humans andother primates, and companion, zoo, and farm animals, including, but notlimited to, cats, dogs, rodents, horses, cows, sheep, pigs, goats, etc.

“Non-immune cell” means a cell that is not normally involved in immuneresponses but that may have the capacity to be modulated by products ofthe immune system.

“SPA” means “synthetic polymeric antigen.” Compound 15 disclosed herein,which is a synthetic peptidoglycan (PG), is a particular SPA. SPAs canbe produced by total synthesis.

“T regulatory cells” or “T_(regs)” refers to a unique lineage ofimmunoregulatory T cells that potently suppress inflammatory effector Tcells in vitro and in vivo. T_(regs) are characterized by expression ofcertain cell surface markers including, for example, CD4 and CD25(CD4+/CD25+).

Natural and Synthetic Polymeric Antigens Useful as Immunomodulators

The structures of CP1, synthetic PG (Compound 15), and a generalizedsynthetic polymeric antigen (SPA) are shown below:

For the present SPAs, “R” represents a substituted amine.

For comparison, the structure of natural peptidoglycan is:

Natural Peptidoglycan

CP1 is a homopolymer of the indicated repeat unit (or one of itssequence isomers). It exists as a distribution of molecular weightscentered around 270 kilodaltons as judged by size exclusionchromatography (dextran as standard). The material is isolated as ahygroscopic white powder that is soluble in water or saline.

SPAs are homopolymers of the indicated general repeat unit structure.These polymers resemble bacterial cell wall peptidoglycans, but areaccessed through chemo-enzymatic total synthesis fromN-acetylglucosamine.

Synthetic PG is an example of an SPA. It is a homopolymer of theindicated repeat unit, existing as a distribution of molecular weightscentered around 150 kilodaltons. The polymer is a hygroscopic whitepowder that is soluble in water or saline.

Natural peptidoglycan in the bacterial cell wall is a single covalentlyclosed macromolecule that precisely defines the shape of a bacterialcell throughout the cell cycle. It is composed of a rigid axis ofparallel polymeric peptidoglycan glycan strands wherein the repeat unitis β-[1,4]-linkedN-acetylglucosaminyl-β-[1,4]-N-acetylmuramylpentapeptide. The glycanstrand is helical in shape with about four repeat units per completeturn of the helix. The more flexible pentapeptide axes extend N to Cfrom the lactyl carboxyls of the muramic acid residues. The peptide isgenerally H₂N-Ala-D-iso-Glu(or iso-Gln)-Lys(or diaminopim-elate,DAP)-D-Ala-D-Ala-COOH (SEQ ID NO: 1). The peptides may be crosslinkedbetween Lys(or DAP) from a donor strand to the carbonyl of thepenultimate D-Ala of an acceptor strand. Although the diagram showscomplete crosslinking for clarity, the actual degree of crosslinking ina living cell varies with genus and is always less than 100%.

In comparison, synthetic PG Compound 15 disclosed herein is linear,i.e., there is no crosslinking in the peptides. In addition, in aminoacid position 2, GABA replaces the naturally occurring D-iso-Glu(D-iso-Gln) residues.

As described below in the method of preparation of CP1 (Example 2), thestarting material for the preparation of CP1 as used herein isobtainable from the American Type Culture Collection (Manassas, Va.) ascrude capsular material from Streptococcus pneumoniae (type 1),originally prepared for production of the Pneumovax vaccine (MerckPharmaceuticals). As described above, CP1 as used in the present studiesis highly purified, and consists solely of the CP1 polysaccharide,without the addition of any other stimulatory antigens or adjuvants. Incontrast, Pneumovax vaccine contains more than 20 capsular typeantigens, and is formulated with an adjuvant. The vaccine is designed tostimulate adaptive immunity and does so effectively. As such, the immuneresponse to this vaccine is completely opposite from that observed whenusing the present isolated, purified CP1 (or Compound 15).

The present inventors have discovered that the bacterial polysaccharidederived from the capsule of Streptococcus pneumoniae (CP1), as well asthe synthetic PG antigen Compound 15 disclosed herein, protect againstthe induction of inflammation in models of intraabdominal abscesses andpost-surgical adhesions. As demonstrated in the examples presentedbelow, investigations into the mechanism of protection induced by thesemolecules reveal that they appear to inhibit the maturation of dendriticcells, the most powerful antigen presenting cells (APCs) in the immunecell repertoire. Immature APCs are unable to activate T cells due to thetheir inability to signal T cells through co-stimulation. Treatment ofhuman PBMCs with either molecule fails to stimulate activation orproliferation of T cells. This is completely unexpected in view of theliterature on both zwitterionic polysaccharides and naturally occurringpeptidoglycans, discussed earlier. Both of these classes of moleculeshave been reported to be mitogens for T cell activation (PCTInternational Publication WO 00/59515; Kalka-Moll et al. (2000) J.Immunol. 164:719-724; Tzianabos et al. (2000) J. Biol. Chem.275:6733-6738; Levinson et al. (1983) Infect. Immun. 39:290-296).Furthermore, CP1 and synthetic PG fail to stimulate Toll-like receptorsin reporter cells in vitro, or to stimulate the expression ofinflammatory cytokines in PBMC cultures, events that would be expectedif maturation of APCs occurs through stimulation of TLR2 or other TLRs(Schwander et al. (1999) J. Biol. Chem. 274:17406-17409; Medzhitov etal. (2001) Nat. Rev. Immunol. 6: 135-145) with subsequent activation ofT cells through the expected cognate interactions between the two cellstypes in the presence of antigen. The present inventors also observe anincrease in the number of CD4+CD25+ cells present in PBMC culturesfollowing treatment with CP1, suggesting that treatment with thismolecule creates a population of immature APCs that drive thestimulation of T regulatory cells within the culture. This hypothesis isfurther supported by functional observations of suppression ofproliferation of T cells in PBMC cultures stimulated with anti-CD3antibodies following treatment with the natural or synthetic polymericantigens. Finally, the inventors have also surprisingly discovered thatwhen human PBMCs are treated in vitro with CP1 or synthetic PG Compound15 as disclosed herein, the response is most notably the expression ofIL10. Negligible expression of IL2, IFN-γ, TNF-α, IL6, or IL12 isobserved. These results are in direct contrast to the body of literatureon the recognition of bacterial polysaccharides by the immune system.Furthermore, the stimulation of an anti-inflammatory response by thesynthetic peptidoglycan polymer disclosed herein is completely novel andunexpected in view of the current body of evidence regarding naturalpeptidoglycans, discussed above, indicating that bacterial peptidoglycanis a potent inflammatory agent. Thus, while natural peptidoglycans areinflammatory, the presently disclosed synthetic peptidoglycan Compound15 is anti-inflammatory. The inventors' surprising discovery of the invitro anti-inflammatory activity of this synthetic peptidoglycancontrasts markedly with previously published observations on theactivity of purified bacterial peptidoglycans, and prompted them to testthe activity of this SPA, as well as CP1, in animal models ofinflammation. As demonstrated below, the inventors observe that boththis synthetic peptidoglycan as well as CP1 exhibit protectivetherapeutic effects in this animal model of inflammation-basedpathology.

Immunomodulatory Activities of Natural and Synthetic Polymeric Antigens(N/S PAs)

The N/S PAs of the present invention induce peripheral blood mononuclearcells (PBMCs) from animals and humans to secrete IL10. IL1 is a type 11cytokine with pleomorphic effects (Moore et al. (2001) Annu. Rev.Immunol. 19:683-765). It has been shown to have potent anti-inflammatoryactivity, down-modulating inflammatory responses of T effector cells(Morel et al. (2002) Immunol. 106:229-236), dendritic cells (Martin etal. (2003) Immunity 18:155-167), and other antigen presenting cells(Williams et al. (2002) J. Leuko. Biol. 72:800-809). IL10 is produced bya variety of cell types, including T cells, dendritic cells, monocytes(Moore et al. (2001) Annu. Rev. Immunol. 19:683-765), and a specializedsub-set of T cells known as T regulatory (Treg) cells (Suri-Payor et al(2001) J. Autoimmun. 16:115-123). In many ways, this cytokine functionsto help maintain a dynamic balance within the immune system. IL10 actsto tamp down unchecked inflammatory responses that could otherwise bedeleterious to the host (Moore et al. (2001) Annu. Rev. Immunol.19:683-765).

Interactions of Natural and Synthetic Polymeric Antigens with DendriticCells

Most microbial antigens signal the immune system through highlyconserved structural motifs referred to as pathogen-associated microbialpatterns (PAMPs) (Medzhitov (2001) Nat. Rev. Immunol. 135-145). PAMPsinteract with Toll-like receptors (TLRs) present on a variety of antigenpresenting cells to initiate a signaling cascade that results in theexpression of pro-inflammatory cytokines such as IL12 and IL6, and avariety of chemokines (Janeway et al. (2002) Annu. Rev. Immunol.20:197-216). Activation of antigen presenting cells through TLRs, inparticular dendritic cells, leads to a maturation process that ischaracterized by increased expression of surface MHC II molecules andco-stimulatory molecules such as CD80 and CD86 (Chakraborty et al.(2000) Clin. Immunol. 94:88-98). This cascade is designed to marshalearly defenders of the innate immune system to respond immediately toinvasion, and forms the basis for the link to long-standing adaptiveimmunity through antigen presentation to T cells (Keller (2001) Immunol.Lett. 78:113-122). Since CP1 is derived from bacterial capsule andsynthetic peptidoglycan Compound 15 is patterned after natural bacterialcell wall-derived peptidoglycan, one might expect that these polymerswould possess PAMPs that could signal through TLRs. Indeed, naturalpeptidoglycan has been shown to be a ligand for TLR2 (Schwandner et al.(1999) J. Biol. Chem. 274:17406-17409). As surprisingly discovered bythe present inventors, N/S PAs do not appear to activate TLR2 or anyother TLR tested in either human or rodent cells. This is furtherevidenced by the lack of expression of IL12, IL6, or otherpro-inflammatory cytokines in PBMC cultures stimulated with N/S PAs. Inaddition, human monocyte-derived dendritic cells are not driven tomaturation by stimulation with N/S PAs. Following treatment with N/SPAs, immature dendritic cells do not demonstrate the characteristicupregulation in MHC II, CD80, or CD86 on their surface, despite the factthat these cells are considered to be the most potent of antigenpresenting cells and avidly internalize these molecules and concentratethem in endocytic vacuoles.

Bacterial lipopolysaccharide (LPS) is a powerful TLR4 agonist (Beulter(2002) Curr. Top Microbiol. Immunol. 270:109-120.), and is commonly usedas a maturation signal for immature dendritic cells (Ardavin et al.(2001) Trends Immunol. 22:691-700). LPS specifically upregulatesco-stimulatory molecules such as CD80 and CD86 on dendritic cells(Michelsen et al. (2001) J. Biol. Chem. 276:25680-25686). These surfacemolecules are essential for signaling T cells to elaborate effectorfunctions such as inflammatory responses. When immature dendritic cellsare co-cultured with N/S PAs and LPS, CD80 and CD86 are not upregulated,suggesting that N/S PAs inhibit the maturation of dendritic cells.

Dendritic Cells

Dendritic cells (DCs) are a family of professional antigen presentingcells that are found in virtually every organ. Dendritic cell subtypeshave been well defined, and it has been demonstrated that these celltypes evolve through several levels of differentiation and maturationthroughout their life span (Jonuleit et al. (2001) Trends in Immunol.22:394-400). Immature dendritic cells are characterized by lowexpression of MHC II molecules, as well as limited expression of theco-stimulatory molecules CD80 and CD86. The expression of these surfacemolecules is dramatically upregulated in response to inflammatorystimuli such as IFNγ or ligation of TLR. Functionally, immature DCs inthe periphery are especially adept at the capture and processing ofantigens. Maturing DCs downregulate these activities, and significantlyupregulate their ability to stimulate naïve T cells through thepresentation of antigen via MHCII and co-stimulation through CD80/86(Banchereau et al (2000) Annu. Rev. Immunol. 18:767-811). Summarized inFIG. 1.

In the absence of inflammation, most peripheral DCs are in an immaturestate, and it is thought that these cells play a major role inmaintenance of peripheral T cell tolerance (recognition of self),induction of T cell anergy, and protection against autoimmunity(Jonuleit et al. (2001) Trends in Immunol. 22:394-400).

The present inventors have observed that treatment of immature dendriticcells with CP1 or synthetic polymeric antigen Compound 15 inhibits theirability to mature, despite the presence of a potent inflammatorystimulus (LPS). The consequences for immune regulation through immatureor semi-mature (low CD80 and CD86 expression) dendritic cells are onlybeginning to be fully appreciated (Lutz et al. (2002) Trends Immunol.23:445-449). It has been suggested that the induction of adaptiveimmunity versus tolerance or suppression of inflammation may bedetermined by the ratio of immature or semi-mature DCs to fully matureDCs in the periphery (Jonuleit et al. (2001) Trends in Immunol.22:394-400; Garza et al. (2000) J. Exp. Med. 191:2021-2028).Chemotherapeutic maintenance of an immature DC population throughtreatment with N/S PAs may inhibit the cognate interactions between Tcells and DCs, thus preventing the clonal expansion of antigen-specificeffector T cells in response to inflammatory stimuli. In view of theentire body of evidence presented herein, however, it is more likelythat the immature DCs generated by N/S PA treatment induce a Tregulatory cell population that directly inhibits the activity ofinflammatory effector T cells, thus affording protection againstinflammatory pathologies. Evidence is mounting in the literature thatimmature DCs induce T regulatory cells in vivo, and further, Tregulatory cells have been induced by immature DCs that specificallyprotect animals from influenza virus infection and prevent rejection inmodels of transplantation (Jonuleit et al. (2001) Trends in Immunol.22:394-400; Dhodapkar et al. (2001) J. Exp. Med. 193:233-238; Thomson etal. (1999) Transplant. Proc. 31:2738-2739). In these studies, immatureDCs were expanded ex vivo and then administered to animals. N/S PAscould provide a unique therapy in which autologous or immunologicallycompatible DCs are rendered chronically immature through ex vivotreatment and then reintroduced into patients to stimulate T regulatoryactivity.

T Regulatory Cells

Recent studies from several laboratories have demonstrated that theimmature dendritic cell is a critical component in the generation of Tregulatory cells (Tregs) (Jonuleit et al. (2001) Trend Immunol.22:394-400). T regulatory cells function to maintain peripheraltolerance, protect against autoimmunity, and participate in modulatinginflammation to allow for appropriate responses to microbial invasion ortissue damage while protecting the host from deleterious bystandereffects (Maloy et al. (2001) Nat. Immunol. 2:816-822).

The most intensely studied Treg phenotype is characterized by theconstitutive expression of the surface markers CD4 and CD25 (Shevach(2002) Nat. Rev. Immunol. 2:389-400). T regulatory cells with thisphenotype have been identified both in vitro and in vivo in both rodents(Taylor et al. (2001) J. Exp. Med. 193:1311-1317) and man (Jonuleit etal. (2001) J. Exp. Med. 193:1285-1294). CD4+CD25+ T cells naturallyoccur in the peripheral circulation at a frequency of approximately2-10% (Shevach (2002) Nat. Rev. Immunol. 2:389-400). During co-cultureof CD4+CD25− target cells with CD4+CD25+ T regulatory cells, the Tregulatory cells inhibit the proliferation of CD4+CD25− target cellsdespite the presence of potent proliferative signals such as antiCD3antibodies or allogeneic APCs (Pasare et al. (2003) Science299:1033-1036). To date, there have been no reports describing adefinitive chemical means to generate T regulatory cells in vivo. Earlystudies reported in the literature indicated that CD4+CD25+ Treg cellsexpressed some IL10 in vitro (Shevach (2002) Nat. Rev. Immunol.2:389-400). Furthermore, in inflammatory models, CD4+ CD25+ cells wereunable to inhibit inflammation in IL10 knockout animals (Shevach (2002)Nat. Rev. Immunol. 2:389-400). These studies led to the widely heldbelief that the mechanism of T regulatory anti-inflammatory activity isvia the expression of IL10. Elegant studies performed in severallaboratories (Jonuleit et al. (2001) J. Exp. Med. 193:1285-1294; Levingset al. (2001) J. Exp. Med. 193:1295-1302; Dieckman et al. (2001) J. Exp.Med. 193:1303-1310) have shown that while CD4+CD25+ T cells do indeedexpress IL10 and/or other cytokines, the mechanism by which theysuppress inflammatory T cells is dependent on cell-cell contact. In theinitial interactions between CD4+CD25+ T cells and their targets,cytokine expression does not play a role. Recently, this seeminglyparadoxical set of observations was clarified by the work of Diekman etal. ((2002) J. Exp. Med. 196:247-253). This group has also shown thatCD4+CD25+ T cells interact with inflammatory T cells through cell-cellcontact. Although the exact nature of the signals transduced by thiscontact is not yet known, these workers demonstrated that one importantconsequence of contact is that the target cells, i.e., CD4+CD25− Tcells, become anergized, and begin to express high levels of IL10. SinceT regulatory cells are relatively rare in the context of the entirety ofthe immune system, this provides a mechanism to amplify theanti-inflammatory effect, and explains the body of data indicating arole for IL10 in systemic anti-inflammation mediated by CD4+CD25+ Tcells.

Human PBMC cultures treated with N/S PAs do not respond by proliferationwhen compared to control cultures treated with polyclonal mitogens suchas phytohaemagglutinin (PHA) or superantigens such as Staphylococcusaureus enterotoxin A (SEA). N/S PAs do, however, stimulate an increasein the percentage of CD4+CD25+ cells present in the culture.Furthermore, when N/S PA-treated PBMC cultures are stimulated with αCD3antibodies, there is a marked suppression in the proliferative capacityof the culture compared to that of untreated controls. Microarrayanalysis further reveals that PBMC cultures treated with N/S PAs andαCD3 antibodies selectively upregulate the expression of IL10 and IL19(an IL10 paralogue) messages in the CD3+ T cell population whiledownregulating several inflammatory cytokine messages such as IL17 andTNFβ.

Taken together, the data disclosed herein suggest that N/S PAs inhibitthe maturation of dendritic cells. Immature dendritic cells have aunique capacity to drive the generation of T regulatory cells. Tregcells may then participate in the inhibition of inflammatory responsesthrough cell-cell signaling as well as through the stimulation of IL10expression from anergized T cells at the sites of inflammation.

IL10

The concept of using recombinant IL10 as an immunotherapeutic is widelyaccepted (Madsen (2002) Gastroenterol. 123:2140-2144; Barnes (2001)Curr. Opin. Allergy Clin. Immunol. 1:555-560; Bremeanu et al (2001) Int.Rev. Immunol. 20:301-331; St. Clair (2000) Curr. Dir. Autoimmun.2:126-149). There are numerous animal models of inflammation in whichIL10 has been shown to be efficacious, e.g., inflammatory bowel disease(IBD), Crohn's disease, rheumatoid arthritis, autoimmune diabetes, andallergic disease (Madsen (2002) Gastroenterol. 123:2140-2144; Barnes(2001) Curr. Opin. Allergy Clin. Immunol. 1:555-560; Bremeanu et al(2001) Int. Rev. Immunol. 20:301-331; St. Clair (2000) Curr. Dir.Autoimmun. 2:126-149). Clinical trials using recombinant IL10 for thetreatment of inflammatory bowel disease have, however, met with mixedresults. Requirements for repeated high dose regimens, as well as someresulting toxicity, have hampered the success of these efforts.Harnessing an individual's immune system to selectively produceendogenous IL10 via T regulatory activity may provide a better route toimmunotherapy. Expression of endogenous IL10, modulated by the hostwithin the entirety of the immune system, may provide the appropriatecontext to achieve efficacy without the requirement for repeated dosingor the problems of cytokine toxicity. Furthermore, the selectiveenhancement of a cell population may prove to be the ideal deliverysystem for such a potent cytokine. Inherent in the immune cellrepertoire is the ability to traffic within the body to sites ofinflammation. An immune cell population that has been given a specifictrafficking signal via a N/S PA-tolerized dendritic cell may populatespecific sites and locally induce IL10 expression. This therapeuticapproach would avoid the problems associated with systemicadministration of potent cytokines and better mimic the naturallylocalized action of this immune mediator.

Intra-Abdominal Abscesses

The formation of intra-abdominal abscesses is the consequence ofcontamination of the peritoneal cavity with colonic bacteria. Thisusually occurs during trauma or surgical interventions. Bacteriastimulate a vigorous inflammatory response, resulting in the recruitmentof macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes,and the release of a variety of inflammatory mediators such as IL1β,TNFα TNFβ, IL17, as well as a number of chemokines (Whal. et al. (1986)J. Exp. Med. 163:884-891; Tzianabos et al. (2002) Curr. Opin. Micro.5:92-95). One possible outcome of this response is the encapsulation ofinvading bacteria by a variety of immune cells interlaced with depositsof fibrin. Once formed, the abscess is relatively resistant toantibiotic therapy, and patients often require surgical intervention todrain the abscess. Although prophylactic antibiotics are given topatients at risk, these interventions are not fully successful. A methodto prevent the initial formation of an abscess by modulation of the hostresponse through T regulatory cell activity and the expression of IL10represents a better form of therapy that could become a standard of carefor at risk surgical procedures.

Post-Surgical Adhesions

Post-surgical adhesions are a significant complication of abdominal,gynecologic, orthopedic, and cardiothoracic surgeries. In the abdomenand pelvic cavity, adhesions are associated with considerable morbidityand can be fatal. In pre-clinical models, exogenously administered IL10has been shown to limit the formation of adhesions (Laan. et al. (1999).J. Immunol. 162:2347-2352; Chung et al. (2002). J. Exp. Med.195:1471-1476). Current therapies in human medicine are, however,designed to interrupt the formation of adhesions after surgical insult.These products involve the introduction of gels or barrier products intothe surgical site. These devices have met with only limited success dueto enhanced infection rates, lack of efficacy, and relatively low ratesof use within the medical community. Better methods to prevent theformation of adhesions are urgently needed.

Like abscess formation, current evidence suggests that the formation ofadhesions also involves activation of inflammatory processes, mostnotably the consistent expression of the inflammatory mediator, IL17,and the deposition of fibrin and other matrix proteins. Together, theseprocesses define a unique intersection between the immune system andpathways of fibrinogenesis and wound repair. Due to the unique nature ofN/S PAs and the potential to manipulate the structures of synthetic PAsand thus their modulating activity, N/S PAs could prove to be usefultools to explore these interactions in greater detail. Specifically, N/SPAs may be useful in the identification and development of biomarkersthat are indicative of specific immune or fibrinogenic responses.

Delayed Type Hypersensitivity Assay for Use as a Clinical StudyBiomarker

In view of the observations that N/S PAs elicit their protective effectsthrough the response of a T regulatory population to inflammatorystimuli, there is a need to develop a specific assay to measure thisactivity for clinical studies. Early phase clinical trials typicallyemploy healthy volunteers for safety and dose response assessment, ascenario that does not necessarily include the induction or measurementof a specific inflammatory pathology. It is therefore necessary todevelop a surrogate biomarker for the activity of these compounds.Delayed Type Hypersensitivity (DTH) reactions in the skin have been usedfor decades to assess exposure to Mycobacterium tuberculosis (TB) inhumans, and more recently to determine the state of T cellresponsiveness in the face of immunocompromise (Anderson et al. (1968)Immunology 15:405-409; Gray et al (1994) Curr. Opin. Immunol. 6:425-437;Kuby et al. (2000) Immunology, W. H. Freeman and Co.) Studies in theliterature have demonstrated that the DTH response is primarily mediatedby T cells and that the inflammatory activity can be adoptivelytransferred to naïve animals by DTH T cells alone (Elices et al. (1993)Clin. Exp. Rheumatol. 11:s77-s80). As disclosed herein, a Guinea pigmodel of DTH has been developed to assess the ability of N/S PAs tolimit the localized inflammatory reaction in the skin. Directmeasurements of the DTH response can be readily observed and measured inhumans and Guinea pigs. Flares, wheals, and/or indurations can beobserved and readily measured quantitatively on the surface of the skin.The antigen used to elicit inflammatory T cell activity in this assay,derived from Candida albicans (Candin), is currently being usedclinically to measure immune competence in individuals undergoingtransplant therapies or suffering from AIDs. This antigen is alsoconsidered to be safer for the general population than TB antigens. Whentested in this model, CP1 demonstrates significant efficacy inpreventing the characteristic skin lesions of DTH. Since it has beenreported in the literature that CD4+CD25+ T regulatory cells areessential components of the memory and protective immunity to C.albicans (Montagnoli et al. (2002) J. Immunol. 169:6298-6308), theseresults provide further evidence that the protective effects of N/S PAsare derived from T regulatory activity.

Mechanism of Action of Naturally Occurring and Synthetic PolymericAntigens: The T Regulatory Cell Hypothesis

The present inventors have conducted detailed investigations into themechanism(s) by which immunomodulatory molecules such as CP1 and thesynthetic polymeric antigen Compound 15 direct and elicitanti-inflammatory effects in mammals, including the induction of Tregulatory cell populations. From these studies, the following picture,summarized in FIG. 2, has emerged.

As depicted in FIG. 2, natural or synthetic immunomodulatory polymericantigens inhibit the maturation of dendritic cells. Immature dendriticcells (iDCs) express low CD80 and CD86 co-stimulatory molecules. In thisstate, iDCs have the unique ability to interact with naïve T cells andinduce the generation of CD4+CD25+ T regulatory cells (pathway B). Inthe face of an inflammatory response, T regulatory cells interact with Teffector cells through cell-cell dependent contact and inhibit theproliferative capacity of these T inflammatory effector cells. Further,contact between T regulatory cells and T effector cells renders theeffectors anergic and stimulates these cells to express large amounts ofIL10. Elicitation of IL10 expression in the former inflammatory T celleffectors serves to amplify the suppressive effects of direct Tregulatory cell contact and broadens the protection against an ongoinginflammatory process. The inhibition of maturation of dendritic cellsobserved by the present investigators could also inhibit the clonalexpansion of T effector cells through the lack of cognate interactionsbetween these two cell types (pathway A). However, the data presentedherein more compellingly support the hypothesis that T regulatory cellsare ultimately generated by the natural or synthetic polymeric antigensof the present invention and afford protection against inflammatorypathologies.

Pharmaceutical Compositions

The natural and synthetic immunomodulatory polymeric antigens disclosedherein can be used to prevent or treat inflammatory pathologies inhumans and other mammals. Thus, in one aspect, the present inventionprovides pharmaceutical compositions for human and veterinary medicaluse comprising CP1 and the synthetic PG of the present invention,together with one or more pharmaceutically or physiologically acceptablecarriers, excipients, or diluents, and optionally, other therapeuticagents. Thus, the present invention also relates to pharmaceuticalcompositions of the presently described immunomodulating polymers incombination with a antibacterial agent or other therapeutic agent, and apharmaceutically acceptable carrier, excipient, or diluent.

The immunomodulatory polymers of the present invention can be deliveredseparately with another anti-bacterial antibiotic drug(s), or in theform of anti-bacterial antibiotic cocktails. An anti-bacterialantibiotic cocktail is a mixture of a molecule of the present inventionand an anti-bacterial antibiotic drug and/or supplementary potentiatingagent. The use of antibiotics in the treatment of bacterial infection isroutine in the art. In this embodiment, a common administration vehicle(e.g., tablet, implant, injectable solution, etc.) can contain both anatural or synthetic polymeric antigen and the anti-bacterial antibioticdrug and/or supplementary potentiating agent. Alternatively, theanti-bacterial antibiotic drug can be separately dosed.

Non-limiting examples of anti-bacterial antibiotic drugs useful in thepresent invention include: penicillin G, penicillin V, ampicillin,arnoxicillin, bacampicillin, cyclacillin, epicillin, hetacillin,pivampicillin, methicillin, nafcillin, oxacillin, cloxacillin,dicloxacillin, flucloxacillin, carbenicillin, ticarcillin, avlocillin,mezlocillin, piperacillin, amdinocillin, cephalexin, cephradine,cefadoxil, cefaclor, cefazolin, cefuroxime axetil, cefamandole,cefonicid, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine,ceftriaxone, moxalactam, cefotetan, cefoperazone, ceftazidme, imipenem,clavulanate, timentin, sulbactam, neomycin, oritavancin, erythromycin,metronidazole, chloramphenicol, clindamycin, lincomycin, vancomycin,trimethoprim-sulfamethoxazole, aminoglycosides, quinolones,tetracyclines, and rifampin. Note Goodman & Gilman's The PharmacologicalBasis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill,New York, (1996) in this regard. The precise amounts of the therapeuticagent used in combination with the immunomodulatory polymers of thepresent invention will depend upon a variety of factors, including thepolymer itself, the dose and dose timing selected, the mode ofadministration, the nature of any surgery that may be contemplated, andcertain characteristics of the subject. Where local administration iscarried out, it will be understood that very small amounts may berequired (nanograms, or possibly picograms). The precise amountsselected can be determined without undue experimentation, particularlysince a threshold amount will be any amount that will favorably enhancesthe desired immune response. A dose in the range of from about onepicogram to about one milligram may be efficacious, depending upon themode of delivery; a dose in the range of from about one nanogram toabout one microgram may also be useful.

Dosing Treatment Regimen, and Administration

The compounds of the present invention can be administered in aneffective amount for inducing protection against a wide variety ofdifferent inflammation-based pathologies, including post-surgicaladhesions and intra-abdominal abscesses associated with bacterialinfection. For such purposes, an effective amount is that amount of acompound of the present invention that will, alone or together withfurther doses or additional therapeutic compounds, inhibit, ameliorate,or prevent the inflammation-based pathology. The dose range can be fromabout one picogram/kilogram bodyweight to about one milligram/kilogrambodyweight, or from about one nanogram/kilogram bodyweight to about onemicrogram/kilogram bodyweight. The absolute amount will depend upon avariety of factors, including the nature of the inflammatory pathologyto be treated, whether the administration is in conjunction withelective surgery or emergency surgery, concurrent treatment, the numberof doses, individual patient parameters including age, physicalcondition, size and weight, and the severity of the inflammation-basedpathology, and can be determined by the medical practitioner with nomore than routine experimentation. It is generally preferred that amaximum dose be used, that is, the highest safe dose according to soundmedical judgment. Multiple doses of the pharmaceutical compositions ofthe invention are contemplated.

Determination of the optimal amount of compound to be administered tohuman or animal patients in need of prevention or treatment of aninflammation-based pathology, as well as methods of administeringtherapeutic or pharmaceutical compositions comprising such compounds, iswell within the skill of those in the pharmaceutical, medical, andveterinary arts. Dosing of a human or animal patient is dependent on thenature of inflammation-based pathology, the patient's condition, bodyweight, general health, sex, diet, time, duration, and route ofadministration, rates of absorption, distribution, metabolism, andexcretion of the compound, combination with other drugs, severity of theinflammation-based pathology, and the responsiveness of the diseasestate being treated, and can readily be optimized to obtain the desiredlevel of effectiveness. The course of treatment can last from severaldays to several weeks or several months, or until a cure is effected oran acceptable diminution or prevention of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient in conjunction with theeffectiveness of the treatment. Persons of ordinary skill can easilydetermine optimum dosages, dosing methodologies, and repetition rates.Optimum dosages can vary depending on the potency of theimmunomodulatory polymeric compound, and can generally be estimatedbased on ED₅₀ values found to be effective in in vitro and in vivoanimal models. Effective amounts of the present compounds for thetreatment or prevention of inflammation-based pathologies, deliveryvehicles containing these compounds, agonists, and treatment protocols,can be determined by conventional means. For example, the medical orveterinary practitioner can commence treatment with a low dose of thecompound in a subject or patient in need thereof, and then increase thedosage, or systematically vary the dosage regimen, monitor the effectsthereof on the patient or subject, and adjust the dosage or treatmentregimen to maximize the desired therapeutic effect. Further discussionof optimization of dosage and treatment regimens can be found in Benetet al., in Goodman & Gilman's The Pharmacological Basis of Therapeutics,Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York, (1996),Chapter 1, pp. 3-27, and L. A. Bauer, in Pharmacotherapy, APathophysiologic Approach, Fourth Edition, DiPiro et al., Eds., Appleton& Lange, Stamford, Conn., (1999), Chapter 3, pp. 21-43, and thereferences cited therein, to which the reader is referred.

In the context of the present invention, the terms “treatment,”“therapeutic use,” or “treatment regimen” as used herein are meant toencompass prophylactic, palliative, and therapeutic modalities ofadministration of the immunomodulatory polymers of the presentinvention, and include any and all uses of the presently claimedcompounds that remedy a disease state, condition, symptom, sign, ordisorder caused by an inflammation-based pathology, or which prevents,hinders, retards, or reverses the progression of symptoms, signs,conditions, or disorders associated therewith. Thus, any prevention,amelioration, alleviation, reversal, or complete elimination of anundesirable disease state, symptom, condition, sign, or disorderassociated with an inflammation-based pathology is encompassed by thepresent invention.

A particular treatment regimen can last for a period of time which mayvary depending upon the nature of the particular inflammation-basedpathology, its severity, and the overall condition of the patient, andmay involve administration of compound-containing compositions from onceto several times daily for several days, weeks, months, or longer.Following treatment, the patient is monitored for changes in his/hercondition and for alleviation of the symptoms, signs, or conditions ofthe disorder or disease state. The dosage of the composition can eitherbe increased in the event the patient does not respond significantly tocurrent dosage levels, or the dose can be decreased if an alleviation ofthe symptoms of the disorder or disease state is observed, or if thedisorder or disease state has been ablated.

An optimal dosing schedule is used to deliver a therapeuticallyeffective amount of the compounds of the present invention. For thepurposes of the present invention, the terms “effective amount” or“therapeutically effective amount” with respect to the compoundsdisclosed herein refers to an amount of compound that is effective toachieve an intended purpose, preferably without undesirable side effectssuch as toxicity, irritation, or allergic response. Although individualpatient needs may vary, determination of optimal ranges for effectiveamounts of pharmaceutical compositions is within the skill of the art.Human-doses can be extrapolated from animal studies (A. S. Katocs,Remington: The Science and Practice of Pharmacy, 19^(th) Ed., A. R.Gennaro, ed., Mack Publishing Co., Easton, Pa., (1995), Chapter 30).Generally, the dosage required to provide a therapeutically effectiveamount of a pharmaceutical composition, which can be adjusted by oneskilled in the art, will vary depending on the age, health, physicalcondition, weight, type and extent of the disease or disorder of therecipient, frequency of treatment, the nature of concurrent therapy (ifany), and the nature and scope of the desired effect(s) (Nies et al.,Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^(th)Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, Chapter3).

Prophylactic modalities for high risk individuals are also encompassedby the present invention. As used herein, the term “high riskindividual” is meant to refer to an individual for whom it has beendetermined, via, e.g., individual or family history or genetic testing,living or working environment or conditions, etc., that there is asignificantly higher than normal probability of being susceptible to aninflammation-based pathology or the onset or recurrence of an associateddisease or disorder. For example, a patient could have a personal and/orfamily medical history that includes frequent occurrences of aparticular disease or disorder. As another example, a patient could havehad such a susceptibility determined by genetic screening according totechniques known in the art (see, e.g., U.S. Congress, Office ofTechnology Assessment, Chapter 5 In: Genetic Monitoring and Screening inthe Workplace, OTA-BA-455, U.S. Government Printing Office, Washington,D.C., 1990, pages 75-99). As part of a treatment regimen for a high riskindividual, the individual can be prophylactically treated to preventinflammation-based pathologies or the onset or recurrence of thedisease, disorder, sign, symptom, or condition. The term“prophylactically effective amount” is meant to refer to an amount of apharmaceutical composition of the present invention that produces aneffect observed as the prevention of infection or inflammation, or theonset or recurrence of a disease, symptom, sign, condition, or disorder.Prophylactically effective amounts of a pharmaceutical composition aretypically determined by the effect they have compared to the effectobserved when a second pharmaceutical composition lacking the activeagent is administered to a similarly situated individual.

For therapeutic use, the immunomodulatory compounds disclosed herein canbe administered to a patient suspected of suffering from aninflammation-based pathology in an amount effective to reduce thesymptomology of the disease, symptom, sign, condition, or disorder. Oneskilled in the art can determine optimum dosages and treatment schedulesfor such treatment regimens by routine methods.

It should be noted that the present invention encompasses the use ofboth CP1 and Compound 15 in combination therapy with one another.

In the case of surgery- or trauma-related abscesses and adhesions, themethods of the present invention can be effectuated by administeringmultiple doses over a three week period preceding surgery, over a twoweek period preceding surgery, over a one week period preceding surgery,when the first dose is administered only 24 hours preceding surgery, andeven when given only after exposure to bacteria. Further doses can beadministered after surgery as well. Any regimen that results in anenhanced immune response to bacterial infection/contamination andsubsequent abscess/adhesion formation can be used, although optimaldoses and dosing regimens are those which would not only inhibit thedevelopment of abscess and/or adhesion formation, but also would resultin a complete protection against abscess or adhesion formation by aparticular bacterial organism or a variety of bacterial organisms.Desired time intervals for delivery of multiple doses of a particularpolymer can be determined by one of ordinary skill in the art employingno more than routine experimentation.

Thus, the present invention is useful whenever it is desirable toprevent bacterial abscess or adhesion formation in a human or animalsubject. This includes prophylactic treatment to prevent such conditionsin planned surgical procedures, as well as in emergency situations.Elective surgeries include the following intraabdominal surgeries: righthemicolectomy; left hemicolectomy; sigmoid colectomy; subtotalcolectomy; total colectomy; laparoscopic or open cholecystectomy;gastrectomy; caesarian section; etc. Emergency surgeries include thoseto correct the following conditions: perforated ulcer (duodenal orgastric); perforated diverticulitis; obstructive diverticulitis; acuteappendicitis; perforated appendicitis; blunt abdominal trauma;penetrating abdominal trauma; second operation to drain abscess; etc.The methods of the present invention are also useful innonintraabdominal surgeries such as cardiac surgeries and surgeries tocorrect wound infections. The present methods are also useful inconnection with diseases that predispose a subject to abscess formationsuch as pelvic inflammatory disease, inflammatory bowel disease, urinarytract infections, and colon cancer. The present methods are thereforeuseful with abscesses of virtually any tissue or organ, includingspecifically, but not limited to, dermal abscesses such as acne. Thoseof ordinary skill in the art to which this invention pertains willreadily recognize the range of conditions and procedures in which thepresent invention is applicable.

In another aspect, the present invention includes a method for inducingprotection against postoperative surgical adhesion formation associatedwith many common types of surgery. The method includes the step ofadministering to a subject in need of such protection a pharmaceuticalpreparation containing an effective amount for reducing postoperativesurgical adhesion formation of the immunomodulating polymer of thepresent invention. It is fully expected that administration of one ormore such polymers at a site separate from the operative site will beeffective in inducing protection against postoperative surgical adhesionformation. This is particularly surprising in view of previousobservations, as discussed above.

PCT International Publication WO 00/59515 teaches that localadministration of certain polymers into the surgical site is effectivefor reducing the incidence of postoperative surgical adhesions. Inaccordance with the present invention, an immunomodulatory polymer canbe effective when given subcutaneously apart from the surgical site atwhich adhesions are likely to form.

The presently disclosed compounds can be administered in an effectiveamount for inducing protection against postoperative surgical adhesionformation. An effective amount for inducing protection againstpostoperative surgical adhesion formation as used herein is that amountof immunomodulating polymer of the present invention that will, alone ortogether with further doses or additional therapeutic compounds, inhibitor prevent the formation of postoperative surgical adhesion. It isbelieved that doses ranging from about one picogram/kilogram bodyweightto about one milligram/kilogram bodyweight, or from about onenanogram/kilogram bodyweight to about one microgram/kilogram bodyweight,will be effective, depending upon the mode of administration. Theabsolute amount will depend upon a variety of factors (including whetherthe administration is in conjunction with elective surgery or emergencysurgery, concurrent treatment, number of doses, and individual patientparameters including age, physical condition, size and weight), and canbe determined via routine experimentation. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment.

Multiple doses of the pharmaceutical compositions of the presentinvention are contemplated for inducing protection against postoperativesurgical adhesion formation. Such multiple doses can be administeredover a three day period beginning on the day preceding surgery. Furtherdoses can be administered post surgery as well. Any regimen that resultsin a reduced postoperative surgical adhesion formation can be used,although optimum doses and dosing regimens are those which would notonly inhibit the development of postoperative surgical adhesionformation, but would also result in complete protection againstpostoperative surgical adhesion formation. Desired time intervals fordelivery of multiple doses of one of the present immunomodulatorypolymers can be determined by one of ordinary skill in the art employingno more than routine experimentation.

Thus, the methods disclosed herein are useful whenever it is desirableto prevent postoperative surgical adhesion formation in a human oranimal subject. This includes prophylactic treatment to prevent adhesionformation following planned surgical procedures, as well as followingemergency operations. Elective surgeries include the followingintraabdominal surgeries: right hemicolectomy; left hemicolectomy;sigmoid colectomy; subtotal colectomy; total colectomy; laparoscopic oropen cholecystectomy; gastrectomy; pancreatectomy; splenectomy; liver,pancreas, small bowel, or kidney transplantation; lysis of adhesions;etc. Emergency intraabdominal surgeries include those to correct thefollowing conditions: perforated ulcer (duodenal or gastric); perforateddiverticulitis; obstructive diverticulitis; bowel obstruction; acuteappendicitis; perforated appendicitis; blunt abdominal trauma;penetrating abdominal trauma; second operation to drain abscess;ruptured abdominal aortic aneurysm, etc. The methods of the presentinvention are also useful in the case of nonintraabdominal surgeriessuch as cardiac surgeries, open and endoscopic orthopedic surgeries,neurosurgeries, gynecologic and pelvic surgeries, and surgeries tocorrect wound infections. The present methods are also useful inconnection with diseases that predispose a subject to spontaneousadhesion formation, such as pelvic inflammatory disease, inflammatorybowel disease, urinary tract infections, and colon cancer. The presentmethods are thus useful with inflammatory processes involving virtuallyany tissue or organ.

When administered to prevent postoperative surgical adhesion formation,the compounds of the present invention can be administered eitherdistant from the operative site, including systemically, or locally intothe operative site at which it is desirable to reduce the likelihood ofpostoperative surgical adhesion formation. The compounds of the presentinvention can be administered as an aqueous solution, as a crosslinkedgel, or as any temporal or physical combination of aqueous solution andcrosslinked gel forms.

The preparations of the present invention can be administered “inconjunction with” infection, meaning close enough in time with thesurgery, trauma, or diseases that predispose the host to abscess oradhesion formation so that a protective effect against abscess oradhesion formation is obtained. The preparations can be administeredlong before surgery in the case of elective surgery (i.e., weeks or evenmonths), preferably with booster administrations closer in time to (andeven after) the surgery. Particularly in emergency situations, thepreparations can be administered immediately before (minutes to hours)and/or after the trauma or surgery. It is important only that thepreparation be administered close enough in time to the surgery so as toenhance the subject's immune response against bacterialinfection/contamination, thereby increasing the chances of a successfulhost response and reducing the likelihood of abscess or adhesionformation.

Those of ordinary skill in the art to which this invention pertains willrecognize that the present methods can be applied to a wide range ofdiseases, symptoms, conditions, signs, disorders, and procedures.Besides abscesses and adhesions, other inflammatory processes andpathologies to which the compounds, compositions, and methods of thepresent invention can be applied include: sepsis; rheumatoid arthritis;myesthenia gravis; inflammatory bowel disease; colitis; systemic lupuserythematosis; multiple sclerosis; coronary artery disease; diabetes;hepatic fibrosis; psoriasis; eczema; acute respiratory distresssyndrome; acute inflammatory pancreatitis; endoscopic retrogradecholangiopancreatography-induced pancreatitis; burns; atherogenesis ofcoronary, cerebral, and peripheral arteries; appendicitis;cholecystitis; diverticulitis; visceral fibrotic disorders (liver, lung,intestinal); wound healing; skin scarring disorders (keloids,hidradenitis suppurativa); granulomatous disorders (sarcoidosis, primarybiliary cirrhosis); asthma; pyoderma gangrenosum; Sweet's syndrome;Behcet's disease; primary sclerosing cholangitis; and cell, tissue, ororgan transplantation.

Formulations

The compounds of the present invention can be administered inpharmaceutically or physiologically acceptable solutions that cancontain pharmaceutically or physiologically acceptable concentrations ofsalts, buffering agents, preservatives, compatible carriers, andoptionally, other therapeutic ingredients. The synthetic PG (Compound15) of the present invention is soluble up to ca. 20 mg/mL in water atneutral pH. Furthermore, aqueous solutions of this compound canaccommodate low (about 0.5 to about 5) weight percentages of glycerol,sucrose, and other such pharmaceutically acceptable excipient materials.CP1 and the SPA Compound 15 disclosed herein can thus be formulated in avariety of standard pharmaceutically acceptable parenteral formulations.

The pharmaceutical compositions of the present invention can contain aneffective amount of CP1 or the presently disclosed SPA, optionallyincluded in a pharmaceutically or physiologically acceptable carrier,excipient, or diluent. The term “pharmaceutically or physiologicallyacceptable carrier, excipient, or diluent” means one or more compatiblesolid or liquid fillers, dilutants, or encapsulating substances that aresuitable for administration to a human or other animal. The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the polymers of the presentinvention, and with each other, in a manner such that there is nointeraction that would substantially impair the desired pharmaceuticalefficiency of CP1 or the SPA.

Compositions suitable for parenteral administration convenientlycomprise sterile aqueous preparations, which can be isotonic with theblood of the recipient. Among the acceptable vehicles and solvents arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed, including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid are useful in the preparation of injectables.Carrier formulations suitable for subcutaneous, intramuscular,intraperitoneal, intravenous, etc. administrations can be found inRemington: The Science and Practice of Pharmacy, 19^(th) Edition, A. R.Gennaro, ed., Mack Publishing Co., Easton, Pa., (1995). CP1 and the SPApolymer of the present invention can be delivered individually, or in amixture comprising the two polymers.

A variety of administration routes are available. The particular modeselected will depend upon whether CP1 or the present SPA is selected,the particular condition being treated, and the dosage required fortherapeutic efficacy. Generally speaking, the methods of the presentinvention can be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofan immune response without causing clinically unacceptable adverseeffects. Preferred modes of administration are parenteral routes. Theterm “parenteral” includes subcutaneous, intravenous, intramuscular, orintraperitoneal injection, or infusion techniques.

The compositions can be conveniently presented in unit dosage form ordosage unit form, and can be prepared by any of the methods well knownin the art of pharmacy. All methods include the step of bringing CP1 orthe SPA into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing CP1 or the SPA into association with aliquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product. CP1 or the SPA can be storedlyophilized.

Other delivery systems can include time-release, delayed-release, orsustained-release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art, includingpolymer-based systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides.

Microcapsules of the foregoing polymers containing drugs are describedin, for example, U.S. Pat. No. 5,075,109. Delivery systems also includenon-polymer systems such as: lipids, including sterols such ascholesterol, cholesterol esters, and fatty acids or neutral fats such asmono-, di-, and tri-glycerides; hydrogel release systems; silasticsystems; peptide-based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5, 736,152, and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5, 407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

The foregoing descriptions provide a comprehensive overview of the manyaspects of the present invention. The following examples illustratevarious aspects thereof and are not intended, nor should they beconstrued, to be limiting thereof in any way.

Example 1

A general procedure for preparing SPA precursor 14 described in PCTInternational Publication Number WO 01/79242. The synthetic approachused herein is outlined in Scheme I and exemplified below.

Definitions DBU 1,8-diazabicyclo[5.4.0]undec-7-ene HOBTN-hydroxybenzotriazole EDCI 1-[3-(dimethyamino)propyl]-3-ethylcarbodiimide hydrochloride Py⁺ protonated pyridine Ac acetyl Phphenyl NHS N-hydroxysuccinimide TLC thin-layer chromatography TFAtrifluoroacetic acid THF tetrahydrofuran DMF dimethyl formamide RP/HPLCreverse-phase HPLC DMAP 4-dimethylaminopyridine NH(TFA) NHC(O)CF₃

General Experimental Conditions

Reactions are carried out with continuous stirring under a positivepressure of nitrogen, except where noted. Reagents and solvents arepurchased and used without further purification, except as noted. TLC isperformed using 0.25 mm silica gel 60 plates from E. Merck with a 254 nmfluorescent indicator. Solvent system specifications are expressed aspercents or ratios by volume. Plates are developed in a covered chamberand visualized by ultraviolet light or by treatment with 5%phosphomolybdic acid in ethanol, with ceric ammonium molybdate inaqueous sulfuric acid, or in the cases of amino acid and peptidederivatives with ninhydrin in acetic acid/n-butanol. All suchvisualization treatments are followed by heating. Flash chromatographyis carried out with silica gel 60, 230-400 mesh (0.040-0.063 mm particlesize) purchased from EM Science or with commercial Biotage prepackaged32-63 mμ KP-Sil cartridges. HPLC analyses and purifications areperformed using Waters X-Terra C8 columns with the specified solventsystem and flow rate.

NMR spectra are reported as chemical shifts in parts-per-million (ppm)downfield from a tetramethylsilane internal standard (0 ppm). ¹H NMRspectra are recorded in the solvent indicated on a Bruker Avancespectrometer at 500.2 MHz, a Varian Mercury spectrometer at 400.21 MHz,or a GE QE-300 spectrometer at 300.2 MHz. Electrospray mass spectra(ES/MS) are recorded on a Micromass Platform LCZ spectrometer. Highresolution mass spectra are recorded on a Micromass QTOF massspectrometer.

Peptide Starting Material

A flask is charged with DMF (117 mL) and compound 1 (20 g, 58.4 mMol).K₂CO₃ (12.1 g, 87.5 mMol) and 1-bromohexane (16.7 mL, 119 mMol) areadded with vigorous stirring and the mixture is heated to 45° C. After 5hr the reaction is complete as evidenced by TLC analysis (25% ethylacetate in hexanes). The mixture is cooled to room temperature anddiluted with ethyl acetate. The solids are filtered under suction andthe filtrate is washed successively with water (1×), N HCl (3×), and0.5M pH 7 buffer (1×). The organic phase is dried (MgSO₄) andconcentrated in vacuo to a thick oil which solidifies on standing. Theoil is taken up in dichloromethane and crystallized from hexanes anddichloromethane. The crystals are dried in vacuo to afford compound 2(24.3 g, 97%). ¹H NMR (400 MHz, CDCl₃) δ 6.51 (br s, 1H), 5.09 (d, 1H,J=7.5 Hz), 4.28 (m, 1H), 4.13 (t, 2H, J=6.8 Hz), 3.37 (m, 2H), 1.82 (m,1H), 1.64 (m, 6H), 1.44 (s, 9H), 1.31 (m, 6H), 0.89 (t, 3H, J=7.0 Hz);ES/MS m/z=427.1 [M+H]⁺, 425.2 [M−H]⁻.

Trifluroracetic acid (62 mL, excess) is added to a stirred solution ofcompound 2 (17.1 g, 13.1 mMol) in dichloromethane (200 mL) at 0° C. Thecooling bath is removed and the reaction mixture is stirred 2 hr atambient temperature. TLC analysis (50% ethyl acetate in hexanes)indicates the absence of starting material. The reaction mixture istransferred to a beaker and layered with water. The pH is adjusted to 9with aqueous NaOH. The organic phase is separated, and the aqueous phaseextracted (2×) with dichloromethane. The organic phase is dried (MgSO₄),and concentrated in vacuo to afford compound 3 as a thick oil (13.7 g,95%), which is taken directly to the coupling step. ES/MS m/z=327.2[M+H]⁺, 325.2 [M−H]⁻.

EDCI {1-[3-(dimethyamino)propyl]-3-ethylcarbodiimide hydrochloride}(7.65 g, 39.9 mMol) is added to a stirred solution ofN-BOC-γ-aminobutyric acid (7.95 g, 39.1 mMol) and NHS (4.59 g, 39.9mMol) in DMF (120 mL) at room temperature. Stirring is continuedovernight, at which time the reaction is judged to be complete by TLCanalysis (10% methanol in chloroform). The crude compound 3 (12.8 g,39.1 mMol) is added in DMF (75 mL), using 5 mL DMF to aid the transfer,followed by diisopropylethyl amine (7.24 mL, 43.0 mMol). After 4 hr thereaction is complete, as judged by TLC analysis (10% methanol inchloroform). The reaction mixture is diluted with ethyl acetate, thenwashed with water (1×), N HCl (2×), and water (1×). The combined aqueousextracts are washed with a single portion of ethyl acetate. The combinedorganic extracts are washed with brine, dried (Na₂SO₄), and concentratedin vacuo. The residue is chromatographed (Biotage 65M; step gradient:1:1 ethyl acetate:hexanes followed by 5% MeOH in ethyl acetate).Appropriate fractions are combined and concentrated in vacuo to affordpure compound 4 (17.7 g, 89%). ES/MS m/z=512.1 [M+H]⁺, 510.1 [M−H]⁻.

Trifluoroacetic acid (5.1 mL, excess) is added to a stirred solution ofcompound 4 (1.69 g, 3.31 mMol) in dichloromethane (33 mL) at 0° C. Thecooling bath is removed and the mixture stirred at ambient temperaturefor 30 min, at which time the reaction is complete as judged by TLCanalysis (10% methanol in chloroform). The dichloromethane solution istransferred to a beaker, layered with water, and the pH adjusted to 9with aqueous NaOH. The organic phase is drawn off and the aqueous phaseis extracted with dichloromethane. The combined organic extracts aredried (Na₂SO₄) and concentrated in vacuo to afford compound 5 as a thickoil (quantitative yield). ES/MS m/z=412.3 [M+H]⁺, 410.2 [M−H]⁻.

Phytanol Phosphate Triethylammonium Salt Starting Material

Phytol 6 (5.0 g, 16.9 mMol) in ethanol (20 mL) is added to a stirredslurry of Raney Ni (about 500 mg) in ethanol (10 mL). The system isbrought under a hydrogen atmosphere at balloon pressure at roomtemperature and stirring is continued overnight. About 50 μL of reactionmixture is removed, filtered through a syringe filter, and concentratedunder a nitrogen stream. ¹H nmr analysis confirms the disappearance ofthe phytol vinylic hydrogen absorption. The reaction mixture is filteredthrough celite and concentrated in vacuo to a yellow oil. The oil isadsorbed on silica gel 60 (10 g) and flash-chromatographed over silicagel 60 (10 g) using a gradient elution (hexanes to 10% ethyl acetate inhexanes). Appropriate fractions are combined and concentrated in vacuoto afford pure phytanol 7 (4.57 g, 90.5%) as a colorless oil. ¹H NMR(400 MHz, CDCl₃) δ 3.68 (m, 2H), 1.46 (m, 25H), 0.74 (m, 15H).

Bis(2,2,2-trichloroethyl)phosphorochloridate (18.7 g, 49.2 mMol) isadded to a stirred solution of compound 7 (9.80 g, 32.8 mMol) and DMAP(802 mg, 6.56 mMol) in dichloromethane. After cooling the solution to 0°C., triethylamine (13.7 mL, 98.4 mMol) is added dropwise via syringe.The cooling bath is removed and the reaction mixture is stirredovernight at ambient temperature, at which point TLC analysis (10% ethylacetate in hexanes) indicates complete reaction. The mixture is thendiluted with dichloromethane, washed with N HCl (3×), and the aqueouslayer is back extracted with dichloromethane. The combined organicextracts are dried (MgSO₄) and concentrated to an oil. The oil ischromatographed over silica gel 60 (50 g) using a gradient of hexanes to15% ethyl acetate in hexanes. Appropriate fractions are combined andconcentrated in vacuo to afford bis(2,2,2-trichloroethyl)phytanylphosphate 8 (20.2 g, 96%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ4.62 (m, 4H), 4.31 (m, 2H), 1.80 (m, 1H), 1.56 (m, 4H), 1.27 (m, 20H),0.86 (m, 15H); ES/MS m/z=641.2 [M+H]⁺, 662.2 [M+Na]⁺.

An HCl/THF solution is prepared by bubbling HCl(g) through anhydrous THFat 0° C. for 2 min. The HCl/THF solution (20 mL) is added portionwise toa stirred suspension of compound 8 (20.1 g, 31.3 mMol) and Zn° dust(24.5 g, 375.6 g-atom) at 0° C. in THF (300 mL). Gas evolution isevident upon each addition. After 1 hr, the Zn° forms small clods in thereaction mixture, and TLC analysis (25% ethyl acetate in hexanes)indicates complete reaction. The reaction mixture is filtered thoroughcelite, concentrated to about ¼ volume, and diluted with ethyl acetate.The organic solution is washed with 1N HCl (3×) and dried (MgSO₄). Afterremoval of the MgSO₄ by suction filtration, triethylamine (4.6 mL, 32.9mMol) is added and the system is then concentrated to a colorless foam.The product is co-distilled with dichloromethane and toluene and thenevacuated (<30 Torr) at room temperature overnight to yield phytanylphosphate triethylamine salt 9. ¹H NMR (400 MHz, CDCl₃) δ 8.74 (m, 1H),5.06 (br, 1H), 4.04 (t, 2H, J=6.5 Hz), 3.28 (dt, 6H, J=9.7, 5.5 Hz),1.68 (m, 1H), 1.52 (m, 2H), 1.38 (t, 9H, J=7.3 Hz), 1.24 (m, 12H), 1.10(m, 9H), 0.86 (m, 15H); ES/MS m/z=377.3 [M−H]⁻.

Lactol Intermediate

The orthogonally-protected disaccharide monopeptide 10 (Saha et al. 2001Organic Lett. 3: 3575) (12.0 g, 12.1 mMol) is added to a stirredsuspension of 10% Pd/C (6.0 g) in 0.23 M HCl in acetic acid (120 mL).The reaction mixture is stirred under an atmosphere of hydrogen (balloonpressure) at 25° C. for 1.5 hr. Analysis of the reaction mixture by TLC(5% MeOH/CHCl₃) shows complete consumption of starting material. Thereaction mixture is filtered through a pad of Celite, concentrated toabout ¼ volume and diluted with methylene chloride. The organic solutionis washed with aqueous NaHCO₃ (×3) and water (×2). The aqueous extractsare combined and extracted with methylene chloride. The combined organiclayers are washed with brine, dried with Na₂SO₄, and concentrated invacuo to afford the lactol product 10a as a white solid (10.2 g, 94% bymass). Inspection of the ¹H NMR spectrum reveals the presence of anunidentified related substance impurity at about 20%. The substance istaken forward with the desired product and removed via purification of asubsequent intermediate. ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, 1H, J=3.4Hz), 7.93 (dt, 2H, J=6.3, 1.7 Hz), 7.70 (dt, 1H, J=7.4, 1.5 Hz), 7.61(m, 2H), 7.40 (t, 1H, J=6.1 Hz), 7.17 (m, 1H), 6.05 (d, 1H, J=9.7 Hz),5.58 (d, 1H, J=3.4 Hz), 5.10 (m, 2H), 2.35 (s, 3H), 2.14 (s, 3H), 2.05(s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.37 (m, 6H); ES/MSm/z=904.4 [M+H]⁺, 902.3 [M−H]⁻.

Monopeptide Dibenzyl Phosphate Intermediate

Compound 10a (13.7 g, 15.2 mMol) in anhydrous dichloromethane (60 mL) isadded rapidly via pressure-equalizing dropping funnel to a vigorouslystirred suspension of tetrazole (4.0 g, 57.8 mMol) and dibenzylN,N′-diethylphosphoramidite (10.4 mL, 29.5 mMol) in anhydrousdichloromethane (40 mL) under argon at 25° C. The reaction mixturebecomes homogeneous within a few minutes. After 2 hr, TLC (5%MeOH/CHCl₃) shows a complete reaction. The mixture is cooled to −78° C.,and 0.2M peracetic acid in methylene chloride (190 mL) is added dropwiseover 10 min with vigorous stirring. After the addition is complete, thecooling bath is removed and the mixture allowed to warm to roomtemperature over 2 hr. TLC (5% MeOH/CHCl₃) shows complete reaction. Themixture is diluted with methylene chloride and extracted: ice-coldsaturated Na₂S₂O₃ (1×), N HCl (1×) and water (1×). The combined aqueousextracts are back-extracted once with methylene chloride. The combinedmethylene chloride solutions are dried over MgSO₄ and concentrated invacuo to a colorless oil. The crude product is adsorbed onpyridine-deactivated silica gel and chromatographed overpyridine-deactivated silica gel using a gradient elution (chloroform to5% methanol in chloroform). Evaporation of solvent provides the puremonophosphate triester 11 (12.4 g, 70%) as a colorless solid. ¹H NMR(400 MHz, CDCl₃) δ 7.88 (d, 2H, J=7.3 Hz), 7.67 (t, 1H, J=7.6 Hz), 7.57(t, 2H, J=7.7 Hz), 7.33 (m, 10H), 7.18 (d, 1H, J=7.4 Hz), 5.98 (m, 2H),5.14 (m, 2H), 5.05 (dd, 2H, J=8.2, 3.1 Hz), 5.00 (d, 2H, J=8.1 Hz) 4.53(m, 2H), 4.37 (m, 3H), 4.29 (dd, 1H, J=12.3, 4.1 Hz), 4.02 (m, 8H), 3.61(d, 1H, J=8.8 Hz), 3.52 (dd, 1H, J=10.9, 8.6 Hz), 3.33 (t, 2H, J=5.8Hz), 2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.95 (s,3H), 1.81 (s, 3H), 1.37 (d, 3H, J=6.4 Hz), 1.33 (d, 3H, J=7.3 Hz); ES/MSm/z=1164.6 [M+H]⁺, 1186.6 [M+Na]⁺, 886.6 [glycosyl]⁺, 1162.6 [M−H]⁻.

Disaccharide Tripeptide Dibenzyl Phosphate Intermediate

DBU (1.30 mL, 8.57 mMol) is added dropwise to a solution ofmonophosphate triester 11 (9.07 g, 7.79 mMol) in dichloromethane (78 mL)under an argon atmosphere. After 15 min, TLC (10% MeOH/CHCl₃) showscomplete consumption of the starting material. The reaction solution isdiluted with dichloromethane and washed twice with 1N HCl. The organiclayer is dried (Na₂SO₄) and concentrated in vacuo, affording the acidanalog of compound 11 as a foam (6.73 g, 87%). ES/MS m/z=1018.6 [M+Na]⁺,718.5 [glycosyl]⁺, 994.6 [M−H]⁻.

The acid analog (2.32 g, 2.33 mMol), peptide 5 (1.05 g, 2.56 mMol) andN-hydroxy-benzotriazole (315 mg, 2.33 mMol) are dissolved in anhydrousDMF (23 mL) at 0° C. EDCI (491 mg, 2.56 mMol) is added and the reactionmixture is stored at −20° C. for 48 hr. Analysis of the reaction mixtureby TLC (15% MeOH/CHCl₃) reveals complete consumption of the startingmaterial. The reaction mixture is concentrated in vacuo, redissolved inethyl acetate, and washed sequentially with water (2×), N HCl (2×),water and brine. The organic solution is dried with MgSO₄ andconcentrated to a foam. The foam is adsorbed on pyridine-deactivatedsilica gel and chromatographed over pyridine-deactivated silica gelusing a gradient of chloroform to 4% methanol in chloroform. Evaporationof solvent affords compound 12 (2.33 g, 72%) as a white solid. ¹H NMR(400 MHz, CDCl₃) δ 7.34 (m, 10H), 6.74 (t, 1H, J=5.9 Hz), 6.45 (d, 1H,J=9.3 Hz), 5.97 (dd, 1H, J=5.4, 3.2 Hz), 5.07 (m, 5H), 2.06 (s, 3H),2.05 (s, 3H), 2.02 (s, 3H), 1.84 (s, 3H), 0.88 (t, 3H, J=7.0 Hz); ES/MSm/z=1389.8 [M+H]⁺, 1412.8 [M+Na]⁺, 1111.7 [glycosyl]⁺, 1387.7 [M−H]⁻.

Disaccharide Tripeptide Phosphate Monopyridyl Salt Intermediate

The disaccharide tripeptide dibenzyl phosphate 12 (7.25 g, 5.22 mMol) inmethanol (30 mL) is added to a suspension of 10% Pd/C (3.63 mg) inmethanol (25 mL), cooled in an ice bath to aid in degassing the reactionsolution. The solution is then warmed to room temperature andhydrogenated at balloon pressure for 2 hr. The catalyst is removed byfiltration through celite and the filtrate is treated with pyridine (1.0mL). The resulting mixture is concentrated to a white solid, which iscollected and dried under high vacuum for 16 hr to afford compound 13(6.32 g, 94%). ¹H NMR (400 MHz, CDCl₃) δ 8.73 (m, 2H), 7.96 (m, 1H),7.56 (m, 2H), 5.88 (m, 1H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),2.01 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 0.88 (t, 3H, J=6.8 Hz).

ES/MS m/z=1209.7 [M+H]⁺, 1232.8 [M+Na]⁺, 1111.8 [glycosyl]⁺, 1207.6[M−H]⁻

Modified Lipid II Intermediate

To a stirred solution of phytanol phosphate triethylamine salt 9 (57 mg,0.118 mMol) in dichloromethane (1.2 mL) at room temperature is addedcarbonyl diimidazole (15 mg, 0.094 mMol). The reaction mixture isstirred overnight. ¹H NMR analysis indicates an 85:15 mixture ofimidazolate derivative and starting phosphate as evidenced by thechemical shifts of the protons on the oxygen-bearing carbon (phosphateat 3.993 ppm, imidazolate at 3.818 ppm). The crude imidazolate isconcentrated in vacuo, taken up in THF to 0.7 mL/mequiv, and used asthus obtained.

The crude imidazolate solution (1.0 mL, 1.5 equiv) is added to asolution of monopyridyl salt 13 (450 mg, 0.349 mMol) in DMF (0.35 mL)and THF (1.5 mL) at room temperature. Vacuum dried 4,5-dicyanoimidazole(103 mg, 0.873 mMol) is added and the reaction mixture stirred at roomtemperature for 18 hr. Imidazolate solution (0.7 mL, 1.0 equiv) is addedand stirring continued for 23 hr. The reaction is complete as evidencedby RP/HPLC analysis:

Waters Xterra C8 analytical column—(4.6 mm×250 mm×5 μm)

Gradient—1:1=50 mM aq. NH₄HCO₃/MeOH to MeOH

Rate—1 mL/min, λ=214 nm, 2 μL reaction mix

Chart—Starting Material @ 10 min, Product @ 20 min

The crude pyrophosphate product is taken on without furthermanipulation.

Aqueous NaOH (1.0N, 4.4 mL) is added to the stirred reaction mixture andstirring is continued while the reaction course is monitored untilstarting material is consumed as evidenced by RP/HPLC (vide supra). Atcompletion, the reaction mixture is diluted with 50 mM aqueous NH₄HCO₃,transferred to a separatory funnel, and extracted with ether (3×). Theaqueous phase is lyophilized to a tan solid (1.15 g). The solid is takenup in minimal 50 mM aq. NH₄HCO₃, passed through a 0.45 μm syringefilter, and chromatographed over a CG-71 resin column (2.2 cm×26 cm, 100mL bed volume) using a nine column volume gradient from 30% MeOH in 50mM aq. NH₄HCO₃ to 100% MeOH. Appropriate 15 mL fracions are pooled,concentrated in a rotary evaporator, and lyophilized to afford puremodified lipid II 15 as a white solid (270 mg, 61%). ¹H NMR (400 MHz,CD₃CN:D₂O=2:1) δ 5.37 (dd, 1H, J=7.1, 3.2 Hz), 4.48 (d, 1H, J=8.3 Hz),4.25 (q, 1H, J=6.7 Hz), 4.15 (m, 1H), 3.46 (m, 1H), 2.89 (t, 1H, J=7.6Hz), 2.22 (t, 1H, J=8.1 Hz), 1.95 (m, 5H), 0.82 (t, 5H, J=6.4 Hz), 0.82(t, 15H, J=6.4 Hz); ES/MS m/z=1221.9 [M+H]⁺, 1219.9 [M−H]⁻.

A 20 mM stock solution of 15 is prepared by dissolving the white powder(370 mg) in water (14.5 mL). To water (79.7 mL) is added PEG 8000 (28.8mL as a 50% stock in water). To this solution is added 0.5M sodiumphosphate buffer at pH 7.0 (5.8 mL), 1M aqueous magnesium chloride (3.6mL). The resulting solution is divided equally among three conicaltubes, and to each is added compound 15 stock solution (4.8 mL) withthorough mixing. The polymerization reaction is initiated by addition of123 μM Staphylococcus aureus MtgA enzyme stock solution (3.9 mL). Thereaction solutions are mixed well and allowed to stand undisturbed for24 hr.

As the polymer forms, it aggregates and settles to the bottom of thetube. The supernatant is removed and centrifuged (3500 rpm, 20 min) torecover any polymer that has been adventitiously removed with thesupernatant. The pellet is dissolved in 0.2M aqueous HCl (5 mL) andtaken on to the next step in this form.

To each of the crude polymer suspensions that remains after decantingthe supernatant is added 5M aqueous HCl (2×100 μL with mixing after eachaddition). The system becomes homogeneous after addition of the acid. Tothe yellowish solutions thus obtained are added the acidified pelletsolutions from processing of the original supernatants (vide supra).These aqueous acidic solutions are incubated at 37° C. overnight, afterwhich the tube contents are pooled to a final volume of about 30 mL. Thesolution is neutralized to pH 7-8 using about 1.2 mL of 5 M aqueousNaOH, at which point the homogeneous solution becomes cloudy. The cloudysolution is centrifuged twice (3500 rpm, 20 min), the pellet beingwashed with water each time and then discarded (final volume of retainedsupernatant=36 mL).

Aqueous 5M NaOH (3.6 mL) is added to bring the final concentration to0.5M. This solution is allowed to stand at room temperature for 2 hr andis then neutralized to pH 6 with 5M aqueous HCl. The solution is dividedinto eight aliquots (8×5 mL, 1×3 mL), each in a 50 mL conical tube. Ninevolumes of ethanol are added to each tube and the solutions are storedovernight in the −20° C. freezer. The tubes are centrifuged (3500 rpm,20 min) and the supernatants carefully removed. After brief drying invacuo, the pellets are dissolved in minimal aqueous NaCl (100 mM) andpooled to a final volume of 16 mL. Nine volumes of ethanol are againadded and the precipitation process repeated. Finally, a third round ofprecipitation is executed.

The final pellet is dissolved in water (40 mL), placed in an AmiconModel 8050 stirred cell concentrator, and subjected toconcentration/dilution cycles until the effluent conductance is nearzero. The solution is then concentrated as much as possible, filteredthrough a pre-washed Millipore Steriflip filter, and lyophilized. Thesynthetic peptidoglycan 15 is thus isolated as a white solid (144 mg,66%).

Verification of Synthetic Peptidoglycan Structure

The structural identity of the synthetic peptidoglycan 15 is determinedby size exclusion chromatography, ¹H NMR spectroscopy, enzymaticsusceptibility and mass spectrometry. Size exclusion chromatography (3.2mm×30 mm Pharmacia Superose 6 column, 20 mM sodium phosphate buffer atpH=7) indicates the midpoint of the size distribution to be about 150kilodaltons based on dextran as standard (range about 75 kD to about 375kD). ¹H NMR (400 MHz, D₂O) δ 4.45 (br s, 1H), 4.32 (br s, 1H), 3.50 (brm, 13H), 2.90 (m, 2H), 2.26 (M, 2H), 1.95 (s, 3H), 1.89 (s, 3H), 1.75(m, 3H), 1.62 (m, 3H), 1.31 (m, 6H).

Synthetic peptidoglycan 15 is rapidly degraded by lysozyme. Bacterialcell wall glycan polymer, a substructure of peptidoglycan, is thenatural substrate for lysozyme. Therefore, lysozyme susceptibilityrepresents prima facie evidence for the glycan substructure of 15.Finally, the lysozyme hydrolysis product of 15,N-acetylgulcos-aminyl-β-[1,4]-N-acetylmuramyl-[Ala-GABA-Lys]-peptide, isconfirmed by ES/MS m/z 781.6 [M+H]⁺, 779.5 [M−H]⁻.

Example 2 Stimulation of IL10 Expression in Human Peripheral BloodMononuclear Cells By Synthetic Bacterial Antigen

Since natural peptidoglycans and bacterial capsular antigens have beenshown to stimulate inflammatory cytokines in vitro and in vivo, wesought to determine the cytokine profile elicited from human peripheralblood mononuclear cells (PBMCs) exposed to either CP1 or synthetic PG.

Preparation of Synthetic PG (Compound 15)

For this and all succeeding examples, Compound 15 is prepared asdescribed in Example 1.

Purification of CP1 From Pneumococcal Polysaccharide Powder (ATCC)

For this and all succeeding examples, CP1 is prepared as follows.

One gram of pneumococcal polysaccharide powder (American Type CultureCollection, Manassas, Va.; lot # 2059900, 5 bottles) is dissolved inpyrogen-free distilled water (ca. 25 mL) with intermittent shaking overthe course of 8 h. The mixture is transferred to the refrigerator (4°C.) and allowed to stand overnight to complete the dissolution and thentransferred to a Teflon bottle. The total volume after quantitativetransfer is ca. 50 mL. Aqueous NaOH (4N, 50 mL) is added and the mixtureis heated for 1 hr at 80° C. with occasional swirling. After cooling toroom temperature, the solution is carefully neutralized with glacialacetic acid (11.3 mL).

The solution is dialyzed (3×, 6-8 kD molecular weight cutoff) againstMilliQ water. After dialysis, the solution is filtered (sterile,pyrogen-free, 0.2μ nylon) and the filtrate is adjusted to 50 mMTris-HCl, pH 8.0. The crude basic hydrolysis product is purified by IEC(ion exchange chromatography; stationary phase=Q-Sepharose Fast Flow,gradient elution mobile phase=50 mM Tris-HCl, pH 8.0 to 50 mM Tris-HCl,pH 8.0 and 11.0M in NaCl). The CP1 containing fractions (180 mL intotal) are identified by analytical SEC (size exclusion chromatography;3.2 mm×30 mm Pharmacia Superose 6 column, 20 mM sodium phosphate bufferat pH 8). The solution is concentrated to 100 mL.

Aqueous NaCl (0.5 M, 200 mL) is added (3× dilution) and the solution isconcentrated to 100 mL using an Amicon concentrator (YM series membrane,10 kD mw cutoff). The NaCl dilution/concentration process is repeated.The resulting solution is diluted with distilled water (200 mL) andconcentrated (Amicon) to 100 mL. This process is repeated five times.The final concentration takes the solution to a volume of 30 mL at whichpoint the conductance of the effluent is <40 μS/cm (1 mM NaCl=10 μS/cm)and the pH=6. After filtration (sterile, pyrogen-free, 0.2μ nylon) andrinsing, the final solution volume is 55 mL. The concentration is ca. 10mg/mL as determined by SEC. The pure CP1 can be stored indefinitely inthis aqueous solution form at 4° C.

The identity of the CP1 is verified from an aliquot by ¹H nmr (Stroop etal. (2002) Carbohydr. Res. 337 335-344) and determined to be free fromtraces of protein and endotoxin by standard assay methodologies.

Human PBMCs are obtained from anonymous donors through the Eli Lilly andCompany donor program. Mononuclear cells are separated by Ficoll-hypaque(Stem Cell Technologies, Vancouver, Canada) sedimentation to eliminatered blood cells and polymorphonuclear leukocytes. The mononuclear layer,consisting of T, B, and mononuclear cells, is cultured in RPMI 1640 with10% fetal bovine serum (Gibco, BRL, Carlsbad Calif.). PBMCs (2×10⁶cells/well) are cultured with several concentrations of Compound 15 orCP1 to determine the optimal response. Although the response to Compound15 or CP1 typically varies among human donors, a concentration of 0.6μg/ml of Compound 15 and 6.0 μg/ml of CP1 gives reproducible andconsistent results and is therefore used in these experiments (FIG. 3).Following isolation, human PBMCs are treated with Compound 15 (0.6μg/ml) or CP1 (6.0 μg/ml) and maintained in culture for eight days.Supernatants are sampled daily and analyzed for cytokine expressionusing a multiplex Enzyme Linked Immunosorbent Assay (Luminex, LincoResearch, St. Charles, Mo.; catalog no. HCYTO-60K). The human multiplexcytokine kits employed in these experiments measure IL1, IL2, IL4, IL6,IL8, IL10, TNFα, and INFγ. In additional experiments, a custom IL12specific antibody bead complex is added to further define the cytokineresponse (Luminex, Linco Research, St. Charles, Mo.). In all assays,results are normalized against untreated media controls. Data areexpressed as the average of triplicate wells±the standard error of theconcentration of cytokines represented. The data represent typicalresults from at least three experiments.

As shown in FIG. 3, in several experiments, the data reveal thattreatment of human PBMCs with Compound 15 or CP1 results in only minimalexpression of most inflammatory cytokines represented in the kit.Surprisingly, the predominant response is the expression of theanti-inflammatory cytokine IL10. The expression of IL10 occurs late inthe time course, detectable at day 5 and continuing to rise at day 8 toconcentrations of approximately 80 pg/ml (Compound 15) to 250 pg/ml(CP1). IL2 and INFγ are only barely detectable early in the time course,whereas the expression of IL4, IL6, IL12 or TNF are not detected at anytime point.

These results suggest that CP1 and synthetic PG selectively induce theexpression of IL10 in PBMC cell cultures, and that they may beefficacious in animal models of inflammation.

Example 3 Interaction of CP1 and Synthetic PG with Toll-Like Receptor 2(TLR2)

Toll-like receptors (TLRs) play a critical role in early innate immunityto invading pathogens by sensing the presence microorganisms within thebody (Akira et al. (2001) Nature Immunol. 2:675-680.) These receptorsrecognize highly conserved structural motifs only expressed by microbialpathogens, called pathogen-associated microbial patterns (PAMPs)(Medzhitov (2001) Nat. Rev. Immunol. 135-145). PAMPs include variousbacterial cell wall components such as lipopolysaccharides (LPS),peptidoglycan and lipopeptides, as well as flagellin, bacterial DNA, andviral double-stranded RNA. Stimulation of TLRs by PAMPs initiates asignaling cascade leading to the activation of the transcription factorNF-KB, which induces the secretion of pro-inflammatory cytokines andeffector cytokines that direct the adaptive immune response (Janeway etal. (2002) Annu. Rev. Immunol. 20:197-216). Since natural peptidoglycanis a PAMP that activates cells via TLR-2 (Iwaki et al. (2002) J. Biol.Chem. 277:24315-24320), we sought to determine if syntheticpeptidoglycan (Compound 15) could also activate NF-κB in vitro. Inaddition, since CP1 behaves in vivo like Compound 15, we investigatedits ability to activate human TLR2 using an NF-κB-reporter assay inHEK293 cells.

These experiments involve transfecting HEK293 cells (American TypeCulture Collection, Manassas, Va.) with two plasmid DNAs. The firstplasmid, called pcDNA3.1/hygrow, contains the human TLR-2 gene. Thesecond plasmid, pNF-κB-luc (Stratagene, La Jolla, Calif.), encodes theNF-κB gene linked to a luciferase reporter gene whose product can befollowed in vitro as a direct measure of NF-κB-activation. To preparethe DNA for transfection into the cells, Fugene6 (Roche, BaselSwitzerland) transfecting reagent is diluted 1:6 in OPTI-MEM(Invitrogen, Carlsbad, Calif.) growth medium. Next, 75 ng of pNF-κB-lucand 300 ng of pcDNA3.1/hygrow DNA are added to the diluted Fugene6 andthe mixture is incubated at 37° C. for 30 minutes. HEK293 cells at aconcentration of 10⁶ cells/ml are added to the DNA/Fugene6 mixture.After gentle mixing, the cell/DNA mixtures are aliquoted into 96 welltissue culture plates at a concentration of 10⁵ cells/well and incubatedfor 24 h at 37° C. in a 5% CO₂ environment. After incubation, varyingconcentrations of test compounds are added to the cells and incubationis allowed to continue for an additional 24 h. The amount of luciferaseactivity resulting from incubation with the compounds is evaluated byremoving the growth media from the cells and replacing it with 100 μl ofRLB lysis solution (Promega, Madison, Wis.). Lysis is completed by asingle freeze/thaw cycle at −80° C. The luciferase activity of each cellculture is determined in a 25 μl aliquot of cell lysate in a VictorLuminometer (Perkin Elmer Life Sciences, Shelton, Conn.) according tothe manufacturer's instructions. A positive control for NF-κB activationin HEK293 cells is incubation of transfected cells with TNFα(Pharmingen, Palo Alto, Calif.) at a concentration of 1 ng/ml.

Table 1 shows that, using varying concentrations ofcommercially-available natural peptidoglycan isolated fromStaphylococcus aureus (Fluka, St. Louis, Mo.), up to 54.5-fold inductionof NFκB activity is observed compared with that of unstimulatedcultures. Another commercially available preparation of peptidoglycanand polysaccharide mixture (PG/PS; Lee Labs Inc., Grayson, Ga.)stimulates up to a 33.7-fold induction of NF-κB in HEK293 cells. Thedata in Table 1 show the lack of NF-κB activation by either Compound 15or CP1 at concentrations up to 500 μg/ml.

TABLE 1 Luciferase Assay for Measurement of TLR2 Activity in HEK293Cells Compound¹: Staphylococcus aureus Concentration peptidoglycan PG/PSCpd 15 (μg/ml) (Fluka) (Lee Labs Inc) CP1 (PG) 500 48.0 33.7 0 0 25051.8 27.0 0 0 125 54.5 15.2 0 0 62.5 50.7 8.8 0 0 31.2 48.6 5.3 0 0 1537.7 3.0 0 0 7.5 34.9 2.6 0 0 3.7 31.8 1.9 0 0 1.8 24.7 1.6 0 0 0.9320.7 1.5 0 0 0.46 17.8 1.0 0 0 ¹Positive stimulation control: culturesincubated with 1 ng/ml TNFα yielded a 22.5-fold increase in luciferaseactivity compared with unstimulated cultures.

These results demonstrate that unlike natural peptidoglycan or bacterialcapsular material (which are PAMPs), CP1 and synthetic PG do not induceactivation of NF-κB through TLR2.

Example 4 Interaction of CP1 and Synthetic PG with Other Toll-LikeReceptors (TLRs)

Concurrently with the studies investigating the interaction of CP1 andsynthetic PG with TLR2, we also tested the interaction of CP1 andsynthetic PG with an expanded list of TLR constructs using the sameNF-κB-reporter assays described above in Example 3 (Table 1). Theresults are shown in Table 2.

TABLE 2 Summary of TLR activation¹ via NFκB using N/S PAs. CompoundEscherichia coli Cpd 15 PG/PS Receptor LPS CP1 (PG) (Lee Labs) TLR2 + −− ++ TLR2/CD14 ++ − − ++ TLR4/CD14 +++ − − − TLR5 + − − − TLR7 +/− − − −TLR8 − − − − ¹ The relative positive activation of NFκB is indicated bythe number of “+” signs while a lack of activation is indicated by a “−”sign.

As shown in Table 2, concentrations of either Compound 15 or CP1 between0.001-100 μg/ml elicit no NF-κB-signaling with any of the other TLRreceptors. In all of these experiments, LPS serves as a positive controlfor TLR4 activation and natural PG serves as a positive control for TLR2activation.

These experiments confirm the previous observation (Example 3) thatneither Compound 15 nor CP1 activates TLR2, even in the presence of anecessary adaptor molecule CD14 (Janeway et al. (2002) Annu. Rev.Immunol. 20:197-216), and extends this observation to five other TLRs.

Example 5 CP1 and Synthetic PG do not Stimulate Maturation of HumanDendritic Cells (DCs)

DCs are often referred to as professional antigen presenting cells andsentinels of the immune system (Banchereau et al. (2000) Annu. Rev.Immunol. 18:767-811). They reside in almost all peripheral tissues in animmature state (iDC), which allows them to phagocytose (or engulf)antigens so they can be processed and presented to the immune system,specifically to naïve T cells (Shortman et al. (2002) Nat. Rev. Immunol.2:151-161). With their cargo of processed antigens, the dendritic cellsmigrate via the blood and lymphatic circulation to lymph nodes, spleen,and other lymphoid tissues. During this journey, they mature, losingtheir ability to take up and process antigen, and begin to display thatantigen on their surfaces. By the time they reach their destinations,they have become potent stimulators of T cells and, with theirmultitentacled (dendritic) shape, proceed to make cell-cell contact withlarge numbers of T cells (Banchereau et al. (2000) Annu. Rev. Immunol18:767-811).

Certain CD (cluster of differentiation) markers, which aresurface-exposed proteins and glycoproteins, can be used to track thematuration state of the dendritic cells (Chakraborty et al. (2000) Clin.Immunol. 94:88-98). Table 3 lists the commonly used CD markers for thispurpose and their relative expression levels on monocytes, immaturedendritic cells (iDC), and mature dendritic cells (mDC) (Chakraborty etal. (2000) Clin. Immunol. 94:88-98).

TABLE 3 Cluster of Differentiation (CD) Markers used to distinguishmonocytes (MO), immature-(iDC) and mature- (mDC) dendritic cells. CellSurface Marker¹: CD1a CD14 CD83 CD86 HLA-DR MO − ++ − − − iDC ++ − − − −mDC ++ − +++ +++ +++ ¹The relative amount of each cell surface marker isindicated in the table by the number of “+” signs while the absence ofthe cell surface marker is indicated by a “−“ sign

Labeling cells with fluorescently-conjugated anti-CD antibodies permitsanalysis of dendritic cell maturation status via determination of meanfluorescence intensity (MFI) of the marker on the surface of a cellpopulation. Flow cytometry is used to analyze large cell samples for thepresence of cell surface markers. In vitro, iDC can be produced byisolating CD14(+) monocytes from human blood and culturing these cellsfor four days with a cocktail of two cytokines (Granulocyte-MacrophageColony-Stimulating Factor (GM-CSF) and Interleukin-4 (IL-4)). Sinceseveral bacterial molecules, for example LPS (Matsunaga et al. (2002)Scand. J. Immunol. 56:593-601) and peptidoglycan (Michelsen et al.(2001) J. Biol. Chem. 276:25680-25686), can induce the differentiationof iDCs to the mDC phenotype (as would occur during activation of theinnate immune system), we were interested in evaluating the potency ofCP1 and synthetic PG in maturing human monocyte-derived dendritic cells.

Human PBMCs are obtained from anonymous donors through the Eli Lilly andCompany donor program. Mononuclear cells are separated by Ficoll-hypaque(Stem Cell Technologies, Vancouver, Canada) sedimentation to eliminatered blood cells and polymorphonuclear leukocytes. The CD14(+) monocytefraction is isolated from PBMCs by incubation with CD14-conjugatedmagnetic beads (Miltenyi Biotech Inc., Auburn, Calif.) followed byphysical separation in a magnetic field using an autoMACS apparatus(Miltenyi Biotech, Inc., Auburn, Calif.). Once isolated, the CD14(+)monocytes are incubated in complete DC media consisting of RPMI 1640containing 10% heat-inactivated Australian fetal bovine serum (FBS), nonessential amino acids, sodium pyruvate, 2-mercaptoethanol,penicillin-streptomycin (as 1× solutions all from Gibco BRL, CarlsbadCalif.). In addition, some cultures are induced to differentiate intoiDCs using complete DC medium containing 20 ng/ml IL-4 (Sigma, St.Louis, Mo.) and 40 ng/ml GM-CSF (Pharmingen, Palo Alto, Calif.) for fourdays at 37° C. with 5% CO₂. After the four day incubation, cells areincubated with CP1, synthetic PG, or LPS for an additional 24 h beforebeing stained for CD marker analysis by flow cytometry. The standardstaining protocol for flow cytometry involves washing the cells twice inDulbecco's phosphate buffered saline (DPBS, Gibco BRL, Carlsbad, Calif.)containing 2% heat inactivated FBS (Gibco BLR, Carlsbad, Calif.) and0.05% sodium azide (Sigma, St. Louis, Mo.), hereafter referred to as“flow wash solution.” After washing, 10⁵ cells/sample are resuspended in100 μl of flow wash solution and 20 μl of pre-dilutedphycoerythrin-conjugated primary anti-CD marker antibody (all antibodiesused are from Pharmingen, Palo Alto, Calif.) for 15 min on ice. Asimilarly conjugated isotype control antibody is included in allanalyses. After incubation, cells are washed three times in flow washsolution. After the final wash, cells are fixed by resuspension in theflow wash solution containing 1% paraformaldehyde (Becton Dickinson,Palo Alto, Calif.). Cell samples are stored at 4° C. and protected fromlight until analysis using an FC500 flow cytometer (Beckman Coulter,Miami, Fla.). Once cells are correctly gated for forward and sidescatter profiles, mean fluorescent intensity (the amount of marker onthe cell surface) is evaluated for 10,000 cells/sample.

The results of these experiments are summarized in Table 4.

TABLE 4 Flow cytometric analysis of monocyte-derived dendritic cellsafter incubation with N/S PAs or LPS. Cell Surface Marker¹: Cell typeCD1a CD14 CD83 CD86 HLA-DR MO 5.1 16.5 5.6 12.8 23.9 iDC 116.1 3.3 7.610.9 7.7 iDC + CP1 122.7 3.3 7.6 11.4 8.7 iDC + Cpd 109.8 3.4 9.5 12.19.1 15 iDC + LPS 124.4 4.1 46.7 75.4 29.2 ¹Numbers represent meanfluorescence intensity of cell surface markers in 10,000 cells/sample.

As shown in Table 4, the panel of surface markers used in thisexperiment confirms that the four day incubation of CD14(+) monocyteswith GM-CSF and IL-4 induces the differentiation of the cells intoimmature dendritic cells (compare the results in Table 4 with theexpected phenotype summarized in Table 3. As shown in Table 4, theseimmature dendritic cells are functionally capable of reaching a maturestate since incubation of these cells with E. coli LPS (the positivecontrol for maturation) significantly increases the staining of CD-83,-86 and HLA-DR on their cell surfaces, which is the expected phenotypeof a mature DC. The data in Table 4 show that incubation with either CP1or Compound 15 fails to change the staining profile from the iDC state,indicating that neither compound is capable of effecting the maturationof dendritic cells.

Example 6 Uptake of Synthetic PG by Immature Human Dendritic Cells(iDCs)

The inhibition of maturation of DCs induced by CP1 and synthetic PG maybe due to the inability of these cells to process these moleculesinternally. Antigen uptake and processing (degradation) are twofundamental properties of APCs (Banchereau et al. (2000) Annu. Rev.Immunol. 18:767-811). DCs are the most potent APCs of the immune systemin part because of their powerful capacity to endocytose or samplematerial from their environment (Shortman et al. (2002) Nat. Rev.Immunol. 2:151-161). To determine whether iDCs are capable ofendocytosing high molecular weight immunomodulatory polysaccharideantigens such as synthetic PG, we prepared a fluorescent derivative ofCompound 15 for use in uptake studies employing confocal microscopy.This imaging technique can be used to localize within cells fluorescentprobes such as the Oregon-green labeled Compound 15 disclosed herein. Inthese experiments, we use as a control molecule fluorescently labeled(FITC) dextran polymer. Dextran (40 kDa in size) is a macromoleculecommonly used for endocytosis experiments (Sallusto et al. (1995) J.Exp. Med. 182:389-400). Since it is a high molecular weight carbohydratepolymer, it is a useful comparator for Compound 15.

Oregon-green labeled Compound 15 is prepared as described in PCTInternational Publication WO 01/79242. Briefly, Oregon-green (MolecularProbes, Eugene, Oreg.)-conjugated Lipid II is included in anMtgA-polymerization reaction at a ratio of 1:4 with unlabeled Lipid 11to produce a 25% Oregon-green labeled polymer. The polymeric material ispurified and treated as previously described. For uptake studies,fluorescent Compound 15 at a final concentration of 50 μg/ml, orLysine-fixable FITC-conjugated dextran (40 kDa size, Molecular Probes,Eugene, Oreg.) at 1 mg/ml, is incubated with human monocyte-derived iDCprepared as described in Example 5 for two minutes at 37° C. Afterincubation, extracellular probe is removed by washing the cells fourtimes in ice cold complete DC medium (Example 5). Washed cells are thenincubated at 37° C. and staining is stopped at two-minute intervals bywashing in 1% paraformaldehyde fix diluted in flow wash solution (whichalso contains the metabolic poison sodium azide; protocol described inExample 5). Glass slide samples are prepared at each time interval andsealed with clear nail polish. Samples are stored at −20° C. andprotected from light until analysis on a Radiance 2100 confocalmicroscope (BioRad Laboratories, Hercules, Calif.).

FIG. 4 shows black and white confocal images of human iDCs treated witheither FITC-Dextran (40 kDa in size) or Oregon-green labeled Compound 15(approx. 150 kDa in size) for two minutes. After incubation with thepolymers, the cells are washed extensively to remove any externalpolymer and the internalized material is followed at two-minuteintervals.

Intracellular localization of either Compound 15 or Dextran is visibleas bright areas in the dark field of the cells after a two-minuteincubation with the polymers (FIG. 4). Furthermore, the internalizedpolymers are not spread throughout the cytoplasm, but are insteadlocalized in discrete packets or vesicles, consistent with theirpresence in endocytic vacuoles.

These results demonstrate that iDCs are capable of endocytosingsynthetic PG.

Example 7 Kinetics of Uptake of Synthetic PG by Immature Human DendriticCells (iDCs)

Since there appears to be such robust uptake of Compound 15 by iDCs(FIG. 4), the fluorescent version of this molecule was used in flowcytometry to visualize the kinetics of polymer uptake.

In these experiments, human monocyte-derived dendritic cells areprepared as described in Example 5. Dendritic cells are resuspended at5×10⁵ cells/sample and incubated on ice at 37° C. At the start of eachtime course, cells are incubated with either fluorescent Compound 15 ata final concentration of 50 μg/ml or Lysine-fixable FITC-conjugateddextran (40 kDa size, Molecular Probes, Eugene, Oreg.) at 1 mg/ml. At 0,2, 10, 20, 30, 40, and 50 minutes after the start of incubation, uptakeis stopped by washing the cells with four washes of ice cold flow washbuffer (Example 5). The washed cells are fixed in paraformaldehyde alsoas described in Example 5. Stained, fixed cells are stored at 4° C.protected from light until analysis using a FC500 flow cytometer(Beckman Coulter, Miami, Fla.). Once cells are correctly gated forforward and side scatter profiles, mean fluorescent intensity of thepopulation is evaluated for 10,000 cells/sample. FIG. 5 shows that overtime, Compound 15 accumulates in the iDC cytoplasm. The same is true forthe control molecule FITC-Dextran. To control for non-specific adhesionof the molecules to the cell surface (which could be read as a positivein this assay), cells are also incubated with fluorescent polymers at 0°C. At this temperature, the iDCs are viable yet unable to endocytosematerial, i.e., they are metabolically inactive (Sallusto et al. (1995)J. Exp. Med. 182:389-400). At this temperature, signal from neither thecontrol molecule (FITC-dextran) nor Compound 15 increases over time(FIG. 5). This indicates that the uptake seen at 37° C. is a result ofcellular endocytosis.

These results demonstrate that iDCs are capable of rapidly endocytosingfluorescently labeled Compound 15, and that the inability of thismolecule to mature DCs is not due to recalcitrance to endocytic uptakethereof.

Example 8 CP1 and Synthetic PG Interfere with LPS-Induced Maturation ofiDCs

As shown above in Table 4 (Example 5), LPS at 50 μg/ml is capable oftransforming iDCs to an mDC phenotype characterized by an increase inco-stimulatory markers (CD83 and CD86) as well as class 11 MajorHistocompatibility (MHC) markers (HLA-DR) (Chakraborty et al. (2000)Clin. Immunol. 94:88-98). We next investigated whether CP1 and syntheticPG are capable of interfering with the transformation of iDCs to mDCs.The results are shown in Table 5.

TABLE 5 Flow cytometric analysis of monocyte-derived dendritic cellsmatured with E. coli LPS in the presence of N/S PAs Cell SurfaceMarker¹: Cell type CD1a CD14 CD83 CD86 HLA-DR iDC + LPS 126.4 4.1 46.775.4 29.2 iDC + LPS + CP1 132.2 4.3 51.1 49.0 31.8 iDC + LPS + Cpd 120.24.1 52.6 59.2 31.9 15 ¹Numbers represent mean fluorescence intensity of10,000 cells/sample.

In these experiments, CD14(+) monocytes are isolated from human PBMCsand differentiated into iDCs as described in Example 5. Afterdifferentiation, iDCs are incubated with either of two known inducers ofcell maturation: E. coli LPS (Matsunaga et al. (2002) Scand. J. Immunol.56:593-601) or a cytokine cocktail containing Tumor Necrosis Factor-α(TNF-α), Interleukin-1β (IL-1β), Prostaglandin E₂, and IL-6 (Dieckman etal. (2002) J. Exp. Med. 196:247-253) for 24 h. To some induced cultureswe also add 50 μg/ml CP 1 or 100 μg/ml Compound 15 at the same time weadd either LPS or cytokines. After incubation, the cells are evaluatedfor CD1a, CD14, CD83, CD86, and HLA-DR expression by flow cytometry asdescribed in Example 5.

In the case of cytokine-matured iDCs, flow cytometry confirms thatmaturation by incubation with the cytokine cocktail occurs; however,incubation with CP1 or synthetic PG has no influence on the maturedphenotype as determined by surface marker analysis (data not shown). Incontrast to this, Table 5 shows that both CP1 and synthetic PG are ableto interfere with LPS-induced maturation of iDCs. Specifically, surfaceexpression of the co-stimulatory marker CD86 is decreased in thepresence of these molecules, while the other markers tested areessentially unchanged. Additional experiments also demonstrate thatCD80, another marker of co-stimulation, is also decreased (data notshown).

The powerful capacity of DCs to activate T cells is linked to theirconstitutive expression of both MHC and costimulatory markers like thefamily B7 markers (i.e., CD80 and CD86) (Banchereau et al. (2000) Annu.Rev. Immunol. 18:767-811). If these molecules are decreased or absentfrom the DC cell surface, the DCs are unable to participate instimulatory cognate interactions with T cells. Schwartz (1990) Science248:1349-1356 was the first to observe that presentation of antigen onMHC molecules in the absence of costimulatory molecules induces T-cellanergy. Thus, DCs can provide both stimulatory (by virtue of being APCs)and downregulatory signals for immune reactions.

To understand fully the significance of the above findings, it isimportant to understand the role of DCs in immune tolerance. Toleranceis an essential property of the immune system whereby self- orauto-antigens do not trigger an immune response (Belz et al. (2002)Immunol. Cell Biol. 80:463-468). Others have shown that when DCs undergoan incomplete maturation (low levels of CD80 and or CD86), or have beentreated with antibodies that block the B7 family of costimulatorymarkers (i.e., CD80 and CD86), these cells can induce antigen-specificunresponsiveness in vitro and T cell anergy in vivo (Lu et al. (1996) J.Immunol. 157:3577-3586; Gao et al. (1999) Immunology 98:159-170).Immature DCs are now understood to contribute to peripheral tolerance byinducing the differentiation of human T regulatory cells (Jonuleit etal. (2000) J. Exp. Med. 192:1213-1222), a group of T cells that displayregulatory functions in vitro and in vivo. Activated T regulatory cellshave also been shown to elicit the production of IL-10, ananti-inflammatory cytokine, through autocrine expression or induction ineffector T cells (Dieckmann et al. (2002) J. Exp. Med. 196:247-253).Thus, the fact that Compound 15 and CP1 appear to influence theexpression of costimulatory markers on the DC surface suggests amechanism of action for these molecules in the induction of toleragenicDCs. These anergic DCs could then induce T-cell anergy directly orthrough the activity of a T regulatory cell population.

Example 9 CP1 and Synthetic PG Are Not Polyclonal Mitopens and Do NotStimulate Proliferation of Lymphocytes in Human PBMC Cultures

Mitogens are substances that nonspecifically induce DNA synthesis andcell division in lymphocytes. LPS is a B-cell specific mitogen (Molleret al. (1973) J. Infect. Dis. 128:52-56), while phytohaemagglutinin(PHA) specifically induces T cells to divide (Boldt et al. (1975) J.Immunol. 114:1532-1536). Peptidoglycan is another T cell mitogen(Levinson et al. (1983) Infect. Immun. 39:290-296). We were thereforeinterested in determining whether CP1 or Compound 15 could stimulatehuman peripheral blood mononuclear lymphocytes (PBMCs) to divide inculture, particularly since Compound 15 is a completely syntheticpeptidoglycan. Cell division is measured in these experiments by uptakeof radiolabeled nucleotide base into the DNA of the proliferating cells.The radioactive counts per minute (cpm) of the culture, measured byscintillation counting, are a direct measure of cellular proliferation.

In this experiment, PBMCs are isolated from a healthy human volunteer asdescribed in Example 2. Isolated PBMCs are aliquoted into round-bottomed96-well tissue culture plates (Falcon Brand, Becton Dickinson, PaloAlto, Calif.) at density of 10⁵ cells/well. Some cells are alsoincubated with 50 μg/ml of CP1, 100 μg/ml Compound 15, or 25 μg/ml PHA(Sigma, St. Louis, Mo.) as a positive control for T cell proliferation.Cells are incubated at 37° C. in a 5% CO₂ atmosphere for up to fourdays. At 30, 54, and 78 hours post inoculation, some cultures are pulsedwith 1 μCi/well of [³H]-thymidine (Specific Activity 6.7 Ci/mmol; ICNInc, Costa Mesa, Calif.) and returned to 37° C. incubation for a further18 hours before being harvested onto filter plates (Packard Instruments,Shelton, Conn.) using a Filtermate harvester (Packard Instruments,Shelton, Conn.). Filterplates are dried after harvesting, prior to theaddition of 20 μl/well of Microscint-O scintillation cocktail (PackardInstruments, Shelton, Conn.). Scintillation counting is performed with aMicroBeta TriLux liquid scintillation counter (Perkin Elmer, Shelton,Conn.).

FIG. 6 shows the typical proliferation response of human PBMCs to thepolyclonal T cell activator PHA. The incorporation of [³H]-thymidineinto PHA-treated cells is close to 100,000 times that of untreated cellsafter two days exposure, and proliferation rates increase up to fourdays. In contrast, neither CP1— nor Compound 15—treated cells respond byDNA proliferation and expansion (FIG. 6). Therefore, these molecules donot appear to behave like polyclonal mitogens in human PBMC cultures.

Example 10 CP1 Stimulates an Increase in CD4+CD25+ T Cells

As CP1 and Compound 15 do not behave like mitogens (Example 9), wehypothesized that the lack of proliferation is due T regulatory cellsuppression. This experiment examines the possibility that thesecompounds stimulate an increase in T regulatory cell numbers as definedby the surface markers CD4 and CD25.

In these experiments, human PBMCs are isolated and cultured at a densityof 10⁵ cells/well in 96-well tissue culture plates as described inExample 2. Some cultures also receive 0.6 or 6.0 μg/ml of CP1immediately after being aliquoted into the tissue culture plates. Cellcultures are incubated at 37° C. in a 5% CO₂ atmosphere for up to sixdays. Each day during culture, cell samples are removed and stained forthe co-expression of CD4 and CD25 on the cell surface by flow cytometry.Samples are stained using the standard staining protocol outlined inExample 5. Antibodies for human CD4, CD25, as well as an isotype controlantibody, are obtained from Pharmingen (Palo Alto, Calif.).

FIG. 7 shows the percentage of CD4+/CD25+ cells in CP1-treated PBMCcultures sampled each day over the course of six days. The percentage ofCD4+/CD25+ cells in untreated PBMCs is highest after two days ofculture, and accounts for up to 2% of total cells in the culture.Incubation of human PBMCs with 0.6 or 6.0 μg/ml CP1 increases thepercentage of CD4+/CD25+ cells to 4.5% and 9.0%, respectively, in thesecultures (FIG. 7).

These data suggest that treatment of human PBMCs with a polysaccharideimmunomodulator such as CP1 induces an increase in the number of cellspossessing a T regulatory phenotype.

Example 11 CP1 and Synthetic PG Suppress the αCD3 Antibody-InducedProliferation of Lymphocytes in Human PBMCs

When an antigen (Ag) is presented to a naïve T cell in the context ofMHCII on the surface of an antigen presenting cell (APC), there isengagement of the MHC-Ag complex with the T cell receptor (TCR)/CD3complex on the surface of the T cell (Weiss et al. (1986) Annu. Rev.Immunol. 4:593-619). This interaction, together with an amplificationsignal generated by CD28-B7 (CD80, CD86) interaction on these two celltypes leads to T cell activation, cytokine stimulation, and celldivision (Weiss et al. (1986) Annu. Rev. Immunol. 4:593-619. In theabsence of Ag or APC, T lymphocytes can become activated and proliferatein vitro by incubation with plate-bound anti-CD antibodies (van Lier etal. (1989) Immunol. 68:45-50). Mimicking the activation by antigens, thebinding of CD3 antibodies to T cells results in the activation oftyrosine kinase, a rise in the intracellular calcium concentration,generation of diacylglycerol, and activation of protein kinase C. Bothcalcium and protein kinase C serve as intracellular messengers for theinduction of gene activation (van Lier et al. (1989) Immunol. 68:45-50).As shown in FIG. 6 of Example 9, anti-CD3 antibody-mediated T cellproliferation is also measured by the incorporation of [³H]-thymidineinto the DNA of dividing cells.

Since proliferation of PBMCs is not observed following treatment witheither CP1 or Compound 15 (FIG. 6), we hypothesized that these moleculesmay stimulate T regulatory cells as suggested by the results shown inFIG. 7. The present experiment is performed to investigate whether thesemolecules induce suppression of lymphocyte proliferation.

In these experiments, human PBMCs are isolated and cultured as describedin Example 2 and plated at 10⁶ cell/ml in T-25 tissue culture flasks(Corning Inc., Corning, N.Y.) for 24 h at 37° C. in a 5% CO₂ atmosphere.Cultures are exposed to either CP1 at 50 μg/ml or PG at 100 μg/ml duringthis period. One day prior to the incubation of cells on antibody coatedplates, anti-human CD3 antibody (Clone UCHT1, Pharmingen, Palo Alto,Calif.) or an isotype-matched control antibody (Pharmingen, Palo Alto,Calif.) is diluted in Dulbecco's phosphate buffered saline (DPBS)(Gibco, BRL, Carlsbad, Calif.), and the wells of a 96-well tissueculture plate are coated with 100 μl aliquots of diluted antibody.Plates are coated overnight at 4° C. and washed three times in DPBSbefore use. Human PBMCs, exposed to CP1, Compound 15, or not exposed toeither compound, are plated into antibody-coated wells at a density of10⁵ cells/well. Tissue culture plates are incubated at 37° C. in a 5%CO₂ atmosphere for 30 or 54 hours before 1 μCi/well of [³H]-thymidine(Specific Activity 6.7 Ci/mmol; ICN Inc, Costa Mesa, Calif.) is added toeach well. Cells are then returned to 37° C. incubation for anadditional 18 h before the cells are harvested as described in Example9. The liquid scintillation counting procedure is also as describedExample 9. The data for this experiment are presented both as raw countsper minute (cpm) of radioactivity and as a stimulation index (SI), whichis the ratio of the cpm of cells in αCD3 antibody-coated wells to thecpm of cells in isotype (control) antibody-coated wells.

FIG. 8 shows that either 48 or 72 hours exposure to αCD3 antibody causeshuman PBMCs to proliferate as shown by the uptake of [³H]-thymidine(FIG. 8, triangles). Furthermore, the amount of proliferation isdirectly correlated to the amount of αCD3 antibody in the well, with thehighest proliferation seen in cells exposed to 0.4 μg/ml αCD3 antibody.FIG. 8 also shows that pre-incubation of human PBMCs with either 50μg/ml of CP1 or 100 μg/ml PG for 24 h prior to incubation with αCD3antibody causes a decrease in the amount subsequent proliferation (FIG.8, diamonds for CP1 and squares for PG Compound 15).

These results demonstrate that N/S PAs inhibit anti-CD3-inducedlymphocyte proliferation.

Example 12 Micro-Array Analysis of Human CD3+ Cells Following Treatmentwith N/S PAs and αCD3 Antibody

The results demonstrating cytokine expression shown in FIG. 3 arecorroborated and extended by measurement of cytokine modulation usingmicroarray technology.

PBMCs are isolated as described in Example 2 and added to 6-well tissueculture plates in a medium containing RPMI with 10% fetal bovine serum(Gibco BRL, Carlsbad, Calif.), 50 μM β-mercaptoethanol, and 500 μg/mlpenicillin/streptomycin (complete medium). T cell density is 2.5×10⁶cells per well. Either 50 μg/ml CP-1,100 μg/ml Compound 15, or completemedium is added to each well of the appropriate plate. Incubation is at37° C. for 24 hours. Simultaneously, 6-well tissue culture plates aretreated with either 0.2 μg/ml αCD3 in sterile Phosphate-Buffered Saline(PBS, Gibco BRL, Carlsbad, Calif.), 5 ml/well, or an equal volume ofsterile PBS. The uninoculated plates are incubated overnight at 4° C.Following incubation, cells treated with CP1 or synthetic PG, oruntreated control cells, are gently resuspended and added to plates thathave either been coated with αCD3 or not, and incubation is continued at37° C. for an additional 48 hours.

PBMCs are then processed with a Pan T Cell Isolation Kit (MiltenyiBiotec, cat. #130-053-001; Auburn, Calif.) in substantial accordancewith the manufacturer's instructions. This kit is a magnetic labelingsystem designed to isolate untouched T cells from peripheral blood.Non-T cells are removed by magnetic separation from unlabeled CD3+ cellsusing an autoMACS (Miltenyi Biotec Inc, Auburn, Calif.). The isolated Tcells are stored at −80° C.

Total RNA is isolated from the cells using Trizol (GibcoBRL, Carlsbad,Calif.) followed by chloroform extraction and subsequent alcoholicprecipitation following procedures specified by the manufacturer. TheRNA is quantitated spectrophotometrically, and its integrity assessed bygel analysis. All RNA preparations are stored at −80° C. until needed.

Total RNA serves as the template for the synthesis of biotin-labeledcDNA. This labeled cDNA is subsequently used as a probe for commerciallyavailable directed microarrays. Specifically, a GEArray Q Series HumanCommon Cytokine Kit, cat. # HS-003N (SuperArray Bioscience Corporation,Frederick, Md.) is employed. Probe synthesis and microarray processingare performed as suggested by the manufacturer. A Typhoon 8600 Imager(Amersham Pharmacia Biotech, Piscataway, N.J.) is used inchemiluminescent mode to capture and store images that are then analyzedusing ImageQuant software (Amersham Pharmacia Biotech, Piscataway,N.J.). Data are exported to Microsoft Excel, and image intensity iscorrected for background and normalized between experiments usingGEArray Analyzer software (SuperArray Bioscience Corporation, Frederick,Md.).

Analysis of the data reveals a cytokine modulation pattern that isconsistent with that seen using the multiplex Enzyme LinkedImmunosorbent Assay as shown in FIG. 3. Table 6 shows that cells exposedto CP1 consistently demonstrate an up-regulation of IL10 and IL19, whichis a homolog of IL10, and a down-regulation of IL17. IL17 is thought tobe expressed mainly by activated T cells, and functions to initiate andmaintain an inflammatory response. Cells that are exposed to αCD3 areactivated and therefore show an up-regulation of IL17, TNF-β, and othercytokines known to participate in the inflammatory process.Anti-CD3-treated cells also show decreases in both IL10 and IL19. Wheneither CP1 or Compound 15 is added to cells that are subsequentlyexposed to αCD3, there is a dramatic increase in IL10 levels,accompanied by concomitant decreases in IL17 and TGF-β.

TABLE 6 Cytokine Response in T Cells Exposed to Various Stimuli StimulusUp-Regulation* Down-Regulation* CP-1 (50 μg/ml) IL19 IL17 α-CD3 (0.2μg/ml) IL17 IL10 TNF-β IL19 α-CD3 (0.2 μg/ml) + IL10** IL17 CP-1 (50μg/ml) IL19 TNF-β *Change from untreated cells of at least 5-fold **Alsoseen with Compound 15 @ 100 μg/ml

The up-regulation of IL10 expression in CD3+ T cells induced by both CP1and Compound 15 in these microarray experiments corroborates the resultsobserved in Example 2, and in animal models, and suggests that thiscytokine can be used as a biological marker to monitor thebiological/immunological activity of both CP1 and synthetic PG in vitroand in vivo. The data also suggest that directed microarrays can be usedto monitor not only the biological activity of the present compounds,but also the biological activity of derivative compounds to determinethe effects of structural differences on immunodulatory potency.

Example 13 N/S PAs Protect Against the Formation of Intra-AbdominalAbscesses

Since CP1 and synthetic PG induce T regulatory cells with suppressivefunction in vitro as well as the late production of IL10 from humanPBMCs (Examples 10 and 11, and Example 2, respectively), we wereinterested in assessing the ability of these polymer antigens to protectanimals against the inflammatory formation of abscesses in vivo. A ratintra-abdominal abscess model is used to address this question.

The rat model of abscess formation employed in these studies is amodification of that described by Onderdonk et al. ((1977) J. Infect.Dis. 136:82-87) and Tzianabos et al. ((1993) Science 262:416-419). MaleLewis rats (Charles River Laboratories, Wilmington, Mass.), weighingbetween 135-175 grams, are used for all experiments. Rats are housed inmicroisolator cages and given chow (Ralston Purina, St. Louis, Mo.) andwater ad libitum. Upon arrival, animals are allowed to acclimate for 24hours. Intra-abdominal abscesses are induced by a single intraperitonealinjection of prepared inoculum containing Bacteroides fragilis (ATCC23745; American Type Culture Collection, Manassas, Va.) (10⁸ colonyforming units per animal) mixed at a 1:6 dilution with an adjuvantsolution containing sterile rat cecal contents. B. fragilis ismaintained at −80° C. in brain heart infusion broth. Cultures are grownanaerobically in brain heart infusion broth to log phase and diluted foruse with rat sterile cecal contents (rSCC). rSCC is prepared from ratcecal pellets that are solubilized in brain heart infusion broth,autoclaved, and then filtered. Animals are euthanized at six dayspost-inoculation and assessed for abscess formation. Animals with one ormore fully formed abscesses are scored as positive. Animals with noabscesses yield a negative score. Individuals scoring the results areblinded to the identity of the experimental groups.

Animals (10 rats/group) are dosed subcutaneously with three doses ofCompound 15 or CP1 at twenty four hour intervals the day before, the dayof, and the day after challenge with B. fragilis/rSCC (Tzianabos et al.J. Clin. Invest. 96:2727 (1995)). Challenge with the inoculum is carriedout by the intraperitoneal route. Animals are administered log dilutionsof Compound 15 or CP1 at 100, 10, and 1 μg (×3)/animal. Results areexpressed as the percent protection (number of animals with noabscesses/treatment group), and statistical significance is calculatedusing the Fishers Exact Probability Test.

As shown in Table 7, both CP1 and Compound 15 produce considerableprotection against the formation of abscesses at both the 100 μg and 10μg doses when compared to that of saline controls. Protection isassessed as the complete absence of abscesses as compared to controlanimals with one or more abscess. Protected animals show no deleteriouseffects of antigen administration, with few, if any, signs of fever orlethargy, which are common symptoms of inflammation. Nor do theseanimals display symptoms of sepsis.

TABLE 7 Activity of N/S PAs in the Rat Abscess Model % of AnimalsAnimals with with % Treatment Group Abscesses/group Abscesses ProtectionCP1 100 μg × 3 SC 1/8 12.5 87.5 CP1 10 μg × 3 SC 2/8 25 75 CP1 1.0 μg ×3 SC 3/8 37.5 62.5 Saline 0.1 ml × 3 SC 6/8 75 25 Cpd 15 100 μg × 3 SC1/8 12.5 87.5 Cpd 15 10 μg × 3 SC 1/8 12.5 87.5 Cpd 15 1.0 μg × 3 SC 2/825 75 Saline 0.1 ml × 3 SC 6/8 75 25

Taken together with the data shown in Examples 2-12, these data suggestthat protection against the inflammatory processes required for theformation of abscesses in response to bacterial challenge in this modelis inhibited by the presence of immature dendritic cells, which candirectly inhibit T cell activation or induce the generation of a Tregulatory population. Direct inhibition of inflammatory cells by Tregulatory cell contact can further stimulate the expression of IL-10.In total, one or more of these events may orchestrate the inhibition ofinflammation seen in the in vivo abscess model.

Example 14 N/S PAs Reduce the Incidence and Severity of Post-SurgicalAdhesions

Exogenous IL10 has been shown to limit the formation of post-surgicaladhesions (Holschneider et al. (1997) J. Surg. Research 70:138-143).Further, T regulatory cells have potent anti-inflammatory activity andhave been shown to limit inflammation in in vivo models (Maloy et al.(2001) Nat. Immunol. 2:816-822; Shevach (2002) Nat. Rev. Immunol.2389-400). T regulatory cells have also been shown to elicit theproduction of IL10 from their target inflammatory T cells (Diekman etal. (2002) J. Exp. Med. 196:247-253). As variously shown in Examples 2,10, 11, 12, and 13, above, CP1 and Compound 15 stimulate the productionof IL10 from PBMCs, an increase in T regulatory cell numbers andfunction in vitro, and afford protection from the formation of abscessesin vivo. Since the inflammatory responses that lead to fibrin depositionand the formation of abscesses is similar to the pathologies involved inadhesion formation, we hypothesized that treatment with CP1 or Compound15 in an adhesion model would likewise stimulate the activity of Tregulatory cells and ultimately the endogenous production of IL10 thatmay result in reduction in the formation of post-surgical adhesions.

To test this hypothesis, male Lewis rats (Charles River Laboratories,Wilmington, Mass.) are dosed subcutaneously with three injections ofCompound 15 or CP1 at twenty four hour intervals the day before, the dayof, and the day after surgical induction of adhesions (Tzianabos et al.(1995) J. Clin. Invest. 96:2727-2731). Rats are administered logdilutions of each compound at 100 μg, 10 μg, and 1 μg (×3) in 0.2 mlsaline/animal. Control groups are administered saline in 0.2 ml volumesat the same dosing schedule. Peritoneal adhesions are induced followingthe methods of Kennedy et al. ((1996) Surgery 120:866-871) and Tzianaboset al. (PCT International Publication WO 00/59515) with minormodifications. Briefly, rats are anesthetized with 2-5% isoflurane inoxygen to a surgical plane of anesthesia. A one to two cm midlineincision is made into the abdominal cavity to expose the cecum. Thececum is aseptically removed from the peritoneal cavity and abraded withsurgical gauze to induce visible microhemorrhages. The cecum is thenre-inserted into the peritoneal cavity. The left and right lateralabdominal walls are inverted aseptically and also abraded in the mannerdescribed above. Following this procedure, 0.2-0.3 ml of rat sterilececal contents (rSCC), prepared as described in Example 13, are added tothe peritoneal cavity as an inflammatory adjuvant (Onderdonk et al.(1982) J. Clin. Invest. 69:9-14). The peritoneum is closed with 3-0 silkfollowed by skin closure with tissue adhesive (3M Animal Care Products,St. Paul, Minn.). Animals are sacrificed one week following surgicalmanipulation and evaluated for the formation of adhesions. Adhesions arescored on a scale of 0-5 using the method described by Kennedy et al((1996) Surgery 120:866-871): 0=no adhesions; 1=thin filmy adhesion;2=more than one thin adhesion; 3=thick adhesion with focal point;4=thick adhesion with planar attachment; and 5=very thick vascularizedadhesions or more than one planar adhesion. This scoring systemapproximates the system used in human medicine, enumerates adhesionspresent, and indicates the severity of the adhesion pathology; higherscores indicate greater severity in inflammation and adhesion formation.The results are shown in Table 8.

TABLE 8 Activity of N/S PAs in the Rat Adhesion Model Range of AdhesionMean Scores/Individual Adhesion Treatment Group Scores Score Median CP1100 μg × 3 SC 0-4 1.8 2.0 (0, 1, 2, 24) CP1 10 μg × 3 SC 1-4 2.4 3.0 (1,1, 3, 3, 4) CP1 1.0 μg × 3 SC 1-4 2.6 3.0 (1, 1, 3, 3, 4) Saline 0.1 ml× 3 SC 3-4 4 4 (3, 3, 4, 5, 5) Cpd 15 100 μg × 3 SC 0-4 1.6 2 (0, 0, 2,2, 4) Cpd 15 10 μg × 3 SC 0-4 2.2 3.0 (0, 1, 3, 3, 4) Cpd 15 1.0 μg × 3SC 0-4 2.2 3 (0, 1, 3, 3, 4) Cpd 15 0.1 μg × 3 SC 1-4 3 3 (1, 3, 3, 4,4) Saline 0.1 ml × 3 SC 3-4 3.6 4 (3, 3, 4, 4, 4)

The data shown in Table 8 demonstrate that adhesion formation in ratstreated with 100 μg of Compound 15 or CP1 is significantly limited(median score=2.0) when compared to that in saline controls (medianscore=4.0). These data demonstrate that these polysaccharide antigenseffectively protect rats from the formation of severe surgically inducedadhesions, and suggest that these polymers induce an anti-inflammatoryeffect in vivo.

Example 15 N/S PAs Inhibit Delayed Type Hypersensitivity Reactions in aGuinea Pig Model

Clinical evaluation of the safety and efficacy of immune modulators suchas CP1 and Compound 15 requires a convenient biomarker. This isnecessary because safety and dose determination are usually determinedin healthy volunteers, where a defined inflammatory process is notmeasured. Furthermore, such a biomarker would be useful in later stagetrials as abscesses and/or adhesions cannot be readily observed andgraded for therapeutic efficacy in a non-invasive manner followingtherapy with immune modulators. Consequently, we developed a delayedtype hypersensitivity (DTH) animal model (Gray et al. (1994) Curr. Opin.Immunol. 6:425-437). This assay can also be used in humans as abiomarker for clinical efficacy studies using the present immunemodulators. Clinically, DTH skin tests are of significant value in theoverall assessment of immunocompetence in humans (Gray et al. (1994)Curr. Opin. Immunol. 6:425-437; Kuby et al. (2000) Immunology, W. H.Freeman and Co). Such tests including the administration of candin asdescribed below are commonly used to test immuno-competence in AIDSpatients.

A Guinea pig model is used to demonstrate the utility of a DTH responseas a biomarker. A localized DTH response in an animal model representsan important source of information with regard to T cell function.Direct measurements of the DTH response can be readily observed andmeasured in humans and animals. Flares, wheals, and/or indurations canbe observed and readily measured quantitatively on the surface of theskin.

For this purpose, female Hartley Guinea pigs (Charles RiverLaboratories, Wilmington, Mass.) weighing 250-299 grams are used for allDTH experiments. Guinea pigs are housed in microisolator cages and givenchow (Ralston Purina, St. Louis, Mo.) and water ad libitum. Uponarrival, the animals are allowed to acclimate for 24 hours. Hair is thenclipped from the back of the animal in an area approximately 2×2 inches.The area is scrubbed with povidone-iodine (H&P Industries/Triad MedicalInc., Mukwonago, Wis.) followed by an alcohol scrub. Next, the animal issensitized to Candida albicans antigens by injecting a 0.2 ml salinesuspension of Candida albicans A26 (ATCC 90234) intradermally on thedorsal side of the neck region. Cultures of Candida albicans A26 aremaintained at −80° C. in a glycerol and lactose freezing solution, andare grown aerobically on Sabourauds and dextrose agar slants (DIFCO,Detroit, Mich.) at 35° C. for 24 hours. Cultures are then suspended insterile saline and adjusted spectrophotometrically to a predeterminedoptical density equivalent to approximately 2.0×10⁷ cells/ml before use.

Three days following sensitization, the animals are treated with theimmunomodulator CP1 formulated in sterile water for injection (AbbottLaboratories, North Chicago, Ill.) at 100, 10 and 1.0 ng per 0.2 ml. Theanimals are injected subcutaneously on the dorsal side of the neck with0.2 ml. A third group of animals dosed with the water vehicle serves asthe positive control group.

Four days following sensitization, the animals are shaved and scrubbedas described above. Four equally spaced areas in the shaved region areinjected intradermally with 0.1 ml of Candin (Allermed Laboratories,Inc., San Diego, Calif.), which serves as a recall antigen for T cellsthat have been previously sensitized to C. albicans. The animals areobserved daily over three days for erythema, wheals, and indurations atthese four sites. Two traverse (vertical and horizontal) diameters ofthe flares are recorded for each site. These are averaged and a mean ofthe flare area (mm²) is calculated. Treated animals are compared tountreated controls in order to assess therapeutic efficacy. The resultsare shown in Table 9.

TABLE 9 The Activity of CP1 in a Model of Delayed Type Hypersensitivity(DTH) Area of Flare (mm²) H₂O CP1 CP1 CP1 Control 1.0 ng × 1 (SC) 10 ng× 1 (SC) 100 ng × 1 (SC) 76.26 ± 5.52 63.34 ± 9.64 50.92 ± 4.26 43.99 ±5.56

The data shown in Table 9 demonstrate a significant reduction in theflare area in animals treated with CP1 as compared to that of controlanimals.

These findings demonstrate that a DTH skin assay is an appropriatebiomarker for clinical use and evaluation of polysaccharideimmunomodulators such as CP1 and synthetic PG Compound 15.

The invention being thus described, it is obvious that the same can bevaried in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

1. A synthetic polymeric antigen having the structure shown in FormulaI:

where n is an integral in the range of from about 375 to about 75, or apharmaceutically acceptable salt thereof.
 2. A composition, comprisingsaid synthetic polymeric antigen or pharmaceutically acceptable saltthereof of claim 1, and a buffer, carrier, diluent, or excipient.
 3. Apharmaceutical composition, comprising said synthetic polymeric antigenor pharmaceutically acceptable salt thereof of claim 1, and apharmaceutically acceptable buffer, carrier, diluent, or excipient.
 4. Asolution comprising said synthetic polymeric antigen or pharmaceuticallyacceptable salt thereof of claim 1, and a solvent.
 5. A method ofinhibiting the maturation of an antigen presenting that is a dendriticcell, comprising contacting in vitro said antigen presenting cell and aneffective amount of the synthetic polymeric antigen or pharmaceuticallyacceptable salt thereof of claim 1, for a time and under conditionseffective to inhibit maturation of said antigen presenting cell.
 6. Themethod of claim 5, wherein inhibition of maturation of said antigenpresenting cell is accompanied by a reduction in the level of expressionof one or more surface markers selected from the group consisting ofCD80 and CD86 by said antigen presenting cell.
 7. The method of claim 5,wherein inhibition of maturation of said antigen presenting cell isaccompanied by a reduction in the level of expression of one or morecytokines selected from the group consisting of IL6, IL12, interferonalpha, and interferon gamma by said antigen presenting cell.
 8. A methodof increasing the expression of interleukin 10 (IL10) in a mammal inneed thereof, comprising: (a) isolating peripheral blood mononuclearcells, or a monocyte-containing fraction thereof, from said mammal; (b)contacting in vitro said isolated peripheral blood mononuclear cells ormonocytes and a composition containing an effective amount of cytokinesthat differentiate monocytes to immature dendritic cells for a time andunder conditions effective to generate immature monocyte-deriveddendritic cells; (c) contacting in vitro said immature monocyte-deriveddendritic cells and an effective amount of the synthetic polymericantigen or pharmaceutically acceptable salt thereof of claim 1, for atime and under conditions effective to prevent maturation of saidimmature monocyte-derived dendritic cells; and (d) administering saidimmature monocyte-derived dendritic cells to said mammal, therebyincreasing the expression of IL10 in said mammal.
 9. The method of claim8, wherein said cytokine composition of step (b) comprisesgranulocyte-macrophage colony-stimulating factor and IL4.